Methods and compositions for plant pest control

ABSTRACT

The present invention provides methods and compositions to improve fungal disease resistance and/or nematode resistance in various crop plants. The present invention also provides for combinations of compositions and methods to improve fungal disease resistance and/or nematode resistance in various crop plants. Powdery mildews are fungal diseases that affect a wide range of plants including cereals, grasses, vegetables, ornamentals, weeds, shrubs, fruit trees, broad-leaved shade and forest trees, that is caused by different species of fungi in the order Erysiphales.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 National Phase of InternationalPatent Application No. PCT/US2013/065561, filed Oct. 18, 2013 andincorporated herein by reference in its entirety, which claims thebenefit of U.S. Provisional Patent Application No. 61/715,549, filedOct. 18, 2012, which is incorporated herein by reference in itsentirety.

INCORPORATION OF SEQUENCE LISTING

A sequence listing containing the file named“MTC58633_PCT_SeqListing.txt”, which is 366,930 bytes (measured inMS-Windows®), contains 213 sequences, and was created on Oct. 14, 2013,is provided herewith and is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Powdery mildews are fungal diseases that affect a wide range of plantsincluding cereals, grasses, vegetables, ornamentals, weeds, shrubs,fruit trees, broad-leaved shade and forest trees, that is caused bydifferent species of fungi in the order Erysiphales. The disease ischaracterized by spots or patches of white to grayish,talcum-powder-like growth that produce tiny, pinhead-sized, sphericalfruiting structures (the cleistothecia or overwintering bodies of thefungus), that are first white, later yellow-brown and finally black. Thefungi that cause powdery mildews are host specific and cannot survivewithout the proper host plant. They produce mycelium (fungal threads)that grow only on the surface of the plant and feed by sendinghaustoria, or root-like structures, into the epidermal cells of theplant. The fungi overwinter on plant debris as cleistothecia or mycelia.In the spring, the cleistothecia produce spores that are moved tosusceptible hosts by rain, wind or insects.

Powdery mildew disease is particularly prevalent in temperate and humidclimates, where they frequently cause significant yield losses andquality reductions in various agricultural settings including greenhouseand field farming. This affects key cereals (e.g. barley and wheat),horticultural crops (e.g. grapevine, pea and tomato) and economicallyimportant ornamentals (e.g. roses). Limited access to natural sources ofresistance to powdery mildews, rapid changes in pathogen virulence andthe time consuming introgression of suitable resistance genes into elitevarieties has led to the widespread use of fungicides to control thedisease. This has, not surprisingly, led to the evolution and spread offungicide resistance, which is especially dramatic amongst the mosteconomically important powdery mildews.

Downy mildew diseases are caused by oomycete microbes from the familyPeronosporaceae that are parasites of plants. Peronosporaceae areobligate biotrophic plant pathogens and parasitize their host plants asan intercellular mycelium using haustoria to penetrate the host cells.The downy mildews reproduce asexually by forming sporangia ondistinctive white sporangiophores usually formed on the lower surface ofinfected leaves. These constitute the “downy mildew” and the initialsymptoms appear on leaves as light green to yellow spots. The sporangiaare wind-dispersed to the surface of other leaves. Depending on thegenus, the sporangia may germinate by forming zoospores or by germ-tube.In the latter case, the sporangia behave like fungal conidia and areoften referred to as such. Sexual reproduction is via oospores.

Most Peronosporaceae are pathogens of herbaceous dicots. Some downymildew genera have relatively restricted host ranges, e.g. Basidiophora,Paraperonospora, Protobremia and Bremia on Asteraceae; Perofascia andHyaloperonospora almost exclusively on Brassicaceae; Viennotia,Graminivora, Poakatesthia, Sclerospora and Peronosclerospora on Poaceae,Plasmoverna on Ranunculaceae. However, the largest genera, Peronosporaand Plasmopara, have very wide host ranges.

In commercial agriculture, downy mildews are a particular problem forgrowers of crucifers, grapes and vegetables that grow on vines.Peronosporaceae of economic importance include Plasmopara viticola whichinfect grapevines, Peronospora tabacina which causes blue mold ontobacco, Bremia lactucae, a parasite on lettuce, and Plasmoparahalstedii on sunflower.

Rusts (Pucciniales, formerly Uredinales) are obligate biotrophicparasites of vascular plants. Rusts affect a variety of plants; leaves,stems, fruits and seeds and is most commonly seen as coloured powder,composed of tiny aeciospores which land on vegetation producingpustules, or uredia, that form on the lower surfaces. During late springor early summer, yellow orange or brown, hairlike or ligulate structurescalled telia grow on the leaves or emerge from bark of woody hosts.These telia produce teliospores which will germinate into aerialbasidiospores, spreading and causing further infection.

In the monocot barley (Hordeum vulgare, Piffanelli et al. Nature. 2004430 (7002):887-91) and the dicots Arabidopsis thaliana (Consonni et al.Nat Genet. 2006 38(6):716-20), tomato (Solanum lycopersicum, Bai et al.Mol Plant Microbe Interact. 2008 21(1):30-9) and pea (Pisum sativum,Humphry et al. Mol Plant Pathol. 2011 Apr. 21. EPUB), loss-of-functionmutations in MLO (Mildew Resistance Locus O) genes confer highlyeffective broad-spectrum powdery mildew resistance. MLO resistanceappears to act early and typically terminates fungal pathogenesis beforeinvasion of the first host cell. The exceptional efficacy and longevityof MLO resistance, has resulted in elite barley lines carryingintrogressed MLO alleles being successfully used in European agriculturefor about three decades. However, MLO mutants have several undesirableagronomic qualities including environment-dependent necrotic leafspotting and reduced yields (Molina-Cano et al. Theor Appl Genet. 2003107(7):1278-87). In addition, barley MLO mutants also show enhancedsusceptibility to the hemibiotroph Magnaporthe grisea and the necrotrophBipolaris sorokinianaare. Lab studies with Arabidopsis powdery resistantMLO mutants suggest that these agronomic defects, including others suchas spontaneous cell wall appositions, cell death, senescence-likechlorosis and enhanced susceptibility to Alternaria alternata, A.brassicicola and Phytophthora infestans (necrotrophic Alternaria spp.and hemibiotrophic P. infestans), respectively, are pleiotropic effectsnot simply linkage drag (Consonni et al. Nat Genet. 2006 38(6):716-20).

Recently a method to increase resistance to soybean rust via thetransgenic knockdown of MLO genes has been disclosed (Markus et al.United States Patent Application 20100192254).

SUMMARY OF THE INVENTION

The present invention provides for compositions comprisingpolynucleotide molecules and methods for treating a plant to alter orregulate gene or gene transcript expression in the plant, for example,by providing RNA or DNA for inhibition of expression. Various aspects ofthe invention provide compositions comprising polynucleotide moleculesand related methods for topically applying such compositions to plantsto regulate endogenous Mildew Resistance Locus O (MLO) genes in a plantcell. The polynucleotides, compositions, and methods disclosed hereinare useful in decreasing levels of MLO transcript and improving fungaldisease and/or nematode resistance of a plant.

In an aspect of the invention, the polynucleotide molecules are providedin compositions that can permeate or be absorbed into living planttissue to initiate localized, partially systemic, or systemic geneinhibition or regulation. In certain embodiments of the invention, thepolynucleotide molecules ultimately provide to a plant, or allow the inplanta production of, RNA that is capable of hybridizing underphysiological conditions in a plant cell to RNA transcribed from atarget endogenous gene or target transgene in the plant cell, therebyeffecting regulation of the endogenous MLO target gene. In certainembodiments, regulation of the MLO target gene, such as by silencing orsuppression of the target gene, leads to the upregulation of anothergene that is itself affected or regulated by decreasing the MLO targetgene's expression.

In certain aspects or embodiments of the invention, the topicalapplication of a composition comprising an exogenous polynucleotide anda transfer agent to a plant or plant part according to the methodsdescribed herein does not necessarily result in nor require theexogenous polynucleotide's integration into a chromosome of the plant.In certain aspects or embodiments of the invention, the topicalapplication of a composition comprising an exogenous polynucleotide anda transfer agent to a plant or plant part according to the methodsdescribed herein does not necessarily result in nor requiretranscription of the exogenous polynucleotide from DNA integrated into achromosome of the plant. In certain embodiments, topical application ofa composition comprising an exogenous polynucleotide and a transferagent to a plant according to the methods described herein also does notnecessarily require that the exogenous polynucleotide be physicallybound to a particle, such as in biolistic mediated introduction ofpolynucleotides associated with a gold or tungsten particles intointernal portions of a plant, plant part, or plant cell. An exogenouspolynucleotide used in certain methods and compositions provided hereincan optionally be associated with an operably linked promoter sequencein certain embodiments of the methods provided herein. However, in otherembodiments, an exogenous polynucleotide used in certain methods andcompositions provided herein is not associated with an operably linkedpromoter sequence. Also, in certain embodiments, an exogenouspolynucleotide used in certain methods and compositions provided hereinis not operably linked to a viral vector.

In certain embodiments, methods for improving fungal disease resistanceand/or nematode resistance in a plant comprising topically applyingcompositions comprising a polynucleotide that suppresses the targetedMLO gene and a transfer agent are provided. In certain embodiments,methods for selectively suppressing the targeted MLO gene by topicallyapplying the polynucleotide composition to a plant surface at one ormore selected seed, vegetative, or reproductive stage(s) of plant growthare provided. Such methods can provide for gene suppression in a plantor plant part on an as needed or as desired basis. In certainembodiments, methods for selectively suppressing the target MLO gene bytopically applying the polynucleotide composition to a plant surface atone or more pre-determined seed, vegetative, or reproductive stage(s) ofplant growth are provided. Such methods can provide for MLO genesuppression in a plant or plant part that obviates any undesired orunnecessary effects of suppressing gene expression at certain seed,vegetative, or reproductive stage(s) of plant development.

In certain embodiments, methods for selectively improving fungal diseaseresistance and/or nematode resistance in a plant by topically applyingthe polynucleotide composition to the plant surface at one or moreselected seed, vegetative, or reproductive stage(s) are provided. Suchmethods can provide for improved fungal disease resistance and/ornematode disease resistance in a plant or plant part on an as needed oras desired basis. In certain embodiments, methods for selectivelyimproving fungal disease and/or nematode resistance in a plant bytopically applying the polynucleotide composition to the plant surfaceat one or more predetermined seed, vegetative, or reproductive stage(s)are provided. Such methods can provide for improving fungal diseaseand/or nematode resistance in a plant or plant part that obviates anyundesired or unnecessary effects of suppressing MLO gene expression atcertain seed, vegetative, or reproductive stage(s) of plant development.

Polynucleotides that can be used to suppress a MLO include, but are notlimited to, any of: i) polynucleotides comprising at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto a transcript of the gene(s) encoding a protein of Table 2 or 3 (SEQID NO:1-27, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78); ii) polynucleotidescomprising at least 18 contiguous nucleotides that are essentiallyidentical or essentially complementary to a MLO or MLO-like gene ofTable 3 comprising a polynucleotide of SEQ ID NO: 29, 31, 33, 35, 37,39, 41, 42, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, or 79; or, polynucleotides comprising at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto a polynucleotide of SEQ ID NO:80-195. In some embodiments, apolynucleotide that comprises a nucleotide sequence that is essentiallyidentical or essentially complementary to at least 18 contiguousnucleotides of SEQ ID NO: 197-213 is provided. In some embodiments, thepolynucleotide comprises a nucleotide sequence that is essentiallyidentical or essentially complementary to SEQ ID NO: 197-213. Methodsand compositions that provide for the topical application of certainpolynucleotides in the presence of transfer agents can be used tosuppress Mildew Resistance Locus O (MLO) gene expression in an optimalmanner. In certain embodiments, the compositions provided herein can beapplied on an “as needed” basis upon scouting for the occurrence offungal disease or nematodes. In certain embodiments, the compositionsprovided herein can be applied as a prophylactic measure to prevent theoccurrence of fungal disease or nematodes. In certain embodiments, thecompositions can be applied in a manner that obviates any deleteriouseffects on yield or other characteristics that can be associated withsuppression of MLO gene expression in a crop plant. The appliedpolynucleotides are complementary to the MLO target host gene in plantsand their topical application leads to suppression of the MLO gene'sactivity.

Provided herein are compositions and methods for controlling plantfungal diseases. Plant fungal diseases that can be controlled with themethods and compositions provided herein include, but are not limitedto, obligate biotrophic powdery mildew, downy mildew and rust fungalinfestations. Certain embodiments relate to methods and compositions forreducing expression of one or more host plant MLO polynucleotide and/orprotein molecules in one or more cells or tissues of the host plant suchthat the host plant is rendered less susceptible to fungal infectionsfrom the order Erysiphales, the family Peronosporaceae, or the orderPucciniales. In certain embodiments, nucleotide and amino acid sequencesof plant Mildew Resistance Locus O (MLO) genes and gene products whichcan be downregulated by methods and compositions provided herein toincrease plant resistance to powdery mildew, downy mildew or rustinfection are disclosed.

Also provided herein are methods and compositions that provide forreductions in expression of targeted MLO polynucleotide and proteinmolecules in at least the cells of a plant root for improved resistanceto nematodes. Nematodes that can be controlled by the methods andcompositions provided herein include, but are not limited to, root knotnematodes (such as Meloidogyne sp.), cyst nematodes (such as Globoderasp. and Heterodera sp.), lesion nematodes (such as Pratylenchus sp.),and the like. In certain embodiments, MLO expression is reduced in plantroot cells from which nematodes feed by providing topically to plantleaves, shoots, roots and/or seeds, compositions comprisingpolynucleotides that comprise at least 18 contiguous nucleotides thatare essentially identical or essentially complementary to a MildewResistance Locus O (MLO) gene or to a transcript of a MLO gene; and atransfer agent.

Also provided are methods and compositions where topically inducedreductions in MLO transcript or protein levels are used to achievepowdery mildew, downy mildew or rust control while minimizingdeleterious pleotropic effects in the host plant. Such methods andcompositions provide for optimized levels of MLO gene inhibition and/oroptimized timing of MLO gene inhibition.

Certain embodiments of the invention are directed to methods forproducing a plant exhibiting an improvement in fungal disease resistanceand/or nematode resistance comprising topically applying to a plantsurface a composition that comprises:

a. at least one polynucleotide that comprises at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto a Mildew Resistance Locus O (MLO) gene or to a transcript of thegene; and

b. a transfer agent, wherein the plant exhibits an improvement in fungaldisease resistance and/or nematode resistance that results fromsuppression of the Mildew Resistance Locus O (MLO) gene. In certainembodiments, the polynucleotide molecule comprises sense ssDNA, sensessRNA, dsRNA, dsDNA, a double stranded DNA/RNA hybrid, anti-sense ssDNA,or anti-sense ssRNA. In certain embodiments, the polynucleotide isselected from the group consisting of SEQ ID NO: 80-195, or wherein thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO: 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, or 79. In some embodiments, a polynucleotide thatcomprises a nucleotide sequence that is essentially identical oressentially complementary to at least 18 contiguous nucleotides of SEQID NO: 197-213 is provided. In some embodiments, the polynucleotidecomprises a nucleotide sequence that is essentially identical oressentially complementary to SEQ ID NO: 197-213. In certain embodiments:(a) the plant is a corn plant, the gene or the transcript is a cornMildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO: 160-162, and SEQ ID NO: 163, or the polynucleotide comprises atleast 18 contiguous nucleotides that are essentially identical oressentially complementary to SEQ ID NO:68-69; (b) the plant is a soybeanplant, the gene or the transcript is a soybean Mildew Resistance Locus O(MLO) gene or transcript, and the polynucleotide molecule is selectedfrom the group consisting of SEQ ID NO: 112-118, and SEQ ID NO: 119, orthe polynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:45 or47, (c) the plant is a cotton plant, the gene or the transcript is acotton Mildew Resistance Locus O (MLO) gene or transcript, and or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to a cotton gene ortranscript that encodes SEQ ID NO:4; (d) the plant is a barley plant,the gene or the transcript is a barley Mildew Resistance Locus O (MLO)gene or transcript, and the polynucleotide molecule is selected from thegroup consisting of SEQ ID NO:80-83, 184, 192, 194, and SEQ ID NO:197-213, or the polynucleotide comprises at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto SEQ ID NO:29; (e) the plant is a cucumber plant, the gene or thetranscript is a cucumber Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO:84-98, and SEQ ID NO:99, or the polynucleotidecomprises at least 18 contiguous nucleotides that are essentiallyidentical or essentially complementary to SEQ ID NO:31, 33, 35, or 37;(f) the plant is a lettuce plant, the gene or the transcript is alettuce Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:100-102, and SEQ ID NO:103, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:39; (g) the plant is a pea plant, the gene orthe transcript is a pea Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO:104-106, and SEQ ID NO:107, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:41; (h)the plant is a Medicago plant, the gene or the transcript is a MedicagoMildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:108-110, and SEQ ID NO:111, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:43; (i) the plant is a pepper plant, the geneor the transcript is a pepper Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO:120-122, and SEQ ID NO:123, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:49; (j)the plant is a tomato plant, the gene or the transcript is a tomatoMildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:124-130, and SEQ ID NO:131, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:51 or 53; (k) the plant is a wheat plant, thegene or the transcript is a wheat Mildew Resistance Locus O (MLO) geneor transcript, and the polynucleotide molecule is selected from thegroup consisting of SEQ ID NO:132-142, and SEQ ID NO:143, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:55, 57or 59; (l) the plant is a grape plant, the gene or the transcript is agrape Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:144-158, and SEQ ID NO:159 or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:61, 63, 65, or 67; (m) the plant is a sorghumplant, the gene or the transcript is a sorghum Mildew Resistance Locus O(MLO) gene or transcript, and the polynucleotide molecule is selectedfrom the group consisting of SEQ ID NO:164-166, and SEQ ID NO:167, orthe polynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:71; or,(n) the plant is a rice plant, the gene or the transcript is a riceMildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:168-182, and SEQ ID NO:183, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:73, 75, 77, or 79. In certain embodiments,the composition comprises any combination of two or more polynucleotidemolecules. In certain embodiments, the polynucleotide is at least 18 toabout 24, about 25 to about 50, about 51 to about 100, about 101 toabout 300, about 301 to about 500, or at least about 500 or moreresidues in length. In certain embodiments, the composition furthercomprises a non-polynucleotide herbicidal molecule, a polynucleotideherbicidal molecule, a polynucleotide that suppresses an herbicidetarget gene, an insecticide, a fungicide, a nematocide, or a combinationthereof. In certain embodiments, the composition further comprises anon-polynucleotide herbicidal molecule and the plant is resistant to theherbicidal molecule. In certain embodiments, the transfer agentcomprises an organosilicone preparation. In certain embodiments, thepolynucleotide is not operably linked to a viral vector. In certainembodiments, the polynucleotide is not integrated into the plantchromosome. Further embodiments of the invention are directed to: aplant made according to any of the above-described methods; progeny ofplants that exhibit the improvements in fungal disease resistance and/ornematode resistance; seed of the plants, wherein seed from the plantsexhibits the improvement in fungal disease resistance and/or nematoderesistance; and a processed product of the plants, the progeny plants,or the seeds, wherein the processed products exhibit the improvement infungal disease resistance and/or nematode resistance. In certainembodiments, the processed product of the plant or plant part exhibitsan improved attribute relative to a processed product of an untreatedcontrol plant and the improved attribute results from the improvedfungal disease resistance and/or nematode resistance. An improvedattribute of a processed product can include, but is not limited to,decreased mycotoxin content, improved nutritional content, improvedstorage characteristics, improved flavor, improved consistency, and thelike when compared to a processed product obtained from an untreatedplant or plant part.

An additional embodiment of the invention is directed to a compositioncomprising a polynucleotide molecule that comprises at least 18contiguous nucleotides that are essentially identical or essentiallycomplementary to a Mildew Resistance Locus O (MLO) gene or transcript ofthe gene, wherein the polynucleotide is not operably linked to apromoter; and, b) a transfer agent. In certain embodiments, thepolynucleotide is selected from the group consisting of SEQ ID NO:80-195, or the polynucleotide comprises at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, or 79. In some embodiments,a polynucleotide that comprises a nucleotide sequence that isessentially identical or essentially complementary to at least 18contiguous nucleotides of SEQ ID NO: 197-213 is provided. In someembodiments, the polynucleotide comprises a nucleotide sequence that isessentially identical or essentially complementary to SEQ ID NO:197-213. In certain embodiments: (a) the gene or the transcript is acorn Mildew Resistance Locus O (MLO) Gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO: 160-162, and SEQ ID NO: 163, or the polynucleotide comprises atleast 18 contiguous nucleotides that are essentially identical oressentially complementary to SEQ ID NO:68-69; (b) the gene or thetranscript is a soybean Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO: 112-118, and SEQ ID NO: 119, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:45 or47; (c) the gene or the transcript is a cotton Mildew Resistance Locus O(MLO) gene or transcript, and the polynucleotide comprises at least 18contiguous nucleotides that are essentially identical or essentiallycomplementary to a cotton gene or transcript that encodes SEQ ID NO:4;(d) the gene or the transcript is a barley Mildew Resistance Locus O(MLO) gene or transcript, and the polynucleotide molecule is selectedfrom the group consisting of SEQ ID NO:80-83, 184, 192, 194, and SEQ IDNO:197-213, or the polynucleotide comprises at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto SEQ ID NO:29; (e) the gene or the transcript is a cucumber MildewResistance Locus O (MLO) gene or transcript, and the polynucleotidemolecule is selected from the group consisting of SEQ ID NO:84-98, andSEQ ID NO:99, or the polynucleotide comprises at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto SEQ ID NO:31, 33, 35, or 37; (f) the gene or the transcript is alettuce Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:100-102, and SEQ ID NO:103, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:39; (g) the gene or the transcript is a peaMildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:104-106, and SEQ ID NO:107, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:41; (h) the gene or the transcript is aMedicago Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:108-110, and SEQ ID NO:111, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:43; (i) the gene or the transcript is apepper Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:120-122, and SEQ ID NO:123, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:49; (j) the gene or the transcript is atomato Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:124-130, and SEQ ID NO:131, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:51 or 53; (k) the gene or the transcript is awheat Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:132-142, and SEQ ID NO:143, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:55, 57 or 59; (l) the gene or the transcriptis a grape Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:144-158, and SEQ ID NO:159 or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:61, 63, 65, or 67; (m) the gene or thetranscript is a sorghum Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO:164-166, and SEQ ID NO:167, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:71; or,(n) the gene or the transcript is a rice Mildew Resistance Locus O (MLO)gene or transcript, and the polynucleotide molecule is selected from thegroup consisting of SEQ ID NO:168-182, and SEQ ID NO:195, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:73, 75,77, or 79. In certain embodiments, the polynucleotide is at least 18 toabout 24, about 25 to about 50, about 51 to about 100, about 101 toabout 300, about 301 to about 500, or at least about 500 or moreresidues in length. In certain embodiments, the composition furthercomprises a non-polynucleotide herbicidal molecule, a polynucleotideherbicidal molecule, a polynucleotide that suppresses an herbicidetarget gene, an insecticide, a fungicide, a nematocide, or a combinationthereof. In certain embodiments, the transfer agent is an organosiliconepreparation. In certain embodiments, the polynucleotide is notphysically bound to a biolistic particle.

Another embodiment of the invention is directed to a method of making acomposition comprising the step of combining at least: (a) apolynucleotide molecule comprising at least 18 contiguous nucleotidesthat are essentially identical or essentially complementary to a MildewResistance Locus O (MLO) gene or transcript of a plant, wherein thepolynucleotide is not operably linked to a promoter or a viral vector;and, (b) a transfer agent. In certain embodiments, the polynucleotide isobtained by in vivo biosynthesis, in vitro enzymatic synthesis, orchemical synthesis. In certain embodiments, the method further comprisescombining with the polynucleotide and the transfer agent at least one ofa non-polynucleotide herbicidal molecule, a polynucleotide herbicidalmolecule, an insecticide, a fungicide, and/or a nematocide. In certainembodiments, the transfer agent is an organosilicone preparation.

Yet another embodiment of the invention is directed to a method ofidentifying a polynucleotide for improving fungal disease resistanceand/or nematode resistance in a plant comprising; (a) selecting apopulation of polynucleotides that are essentially identical oressentially complementary to a Mildew Resistance Locus O (MLO) gene ortranscript of a plant; b) topically applying to a surface of at leastone of the plants a composition comprising at least one polynucleotidefrom the population and an transfer agent to obtain a treated plant;and, c) identifying a treated plant that exhibits suppression of theMildew Resistance Locus O (MLO) gene or exhibits an improvement infungal disease resistance or exhibits an improvement in nematoderesistance, thereby identifying a polynucleotide that improves fungaldisease resistance and/or nematode resistance in the plant. In certainembodiments, the polynucleotide is selected from the group consisting ofwherein the polynucleotide is selected from the group consisting of SEQID NO: 80-195, or wherein the polynucleotide comprises at least 18contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, or 79. In someembodiments, a polynucleotide that comprises a nucleotide sequence thatis essentially identical or essentially complementary to at least 18contiguous nucleotides of SEQ ID NO: 197-213 is provided. In someembodiments, the polynucleotide comprises a nucleotide sequence that isessentially identical or essentially complementary to SEQ ID NO:197-213. In certain embodiments: (a) the plant is a corn plant, the geneor the transcript is a corn Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO: 160-162, and SEQ ID NO: 163, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:68-69;(b) the plant is a soybean plant, the gene or the transcript is asoybean Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO: 112-118, and SEQ ID NO: 119, or the polynucleotide comprises atleast 18 contiguous nucleotides that are essentially identical oressentially complementary to SEQ ID NO:45 or 47, (c) the plant is acotton plant, the gene or the transcript is a cotton Mildew ResistanceLocus O (MLO) gene or transcript, and or the polynucleotide comprises atleast 18 contiguous nucleotides that are essentially identical oressentially complementary to a cotton gene or transcript that encodesSEQ ID NO:4; (d) the plant is a barley plant, the gene or the transcriptis a barley Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:80-83, 184, 192, 194, and SEQ ID NO: 197-213, or the polynucleotidecomprises at least 18 contiguous nucleotides that are essentiallyidentical or essentially complementary to SEQ ID NO:29; (e) the plant isa cucumber plant, the gene or the transcript is a cucumber MildewResistance Locus O (MLO) gene or transcript, and the polynucleotidemolecule is selected from the group consisting of SEQ ID NO:84-98, andSEQ ID NO:99, or the polynucleotide comprises at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto SEQ ID NO:31, 33, 35, or 37; (f) the plant is a lettuce plant, thegene or the transcript is a lettuce Mildew Resistance Locus O (MLO) geneor transcript, and the polynucleotide molecule is selected from thegroup consisting of SEQ ID NO:100-102, and SEQ ID NO:103, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:39; (g)the plant is a pea plant, the gene or the transcript is a pea MildewResistance Locus O (MLO) gene or transcript, and the polynucleotidemolecule is selected from the group consisting of SEQ ID NO:104-106, andSEQ ID NO:107, or the polynucleotide comprises at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto SEQ ID NO:41; (h) the plant is a Medicago plant, the gene or thetranscript is a Medicago Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO:108-110, and SEQ ID NO:111, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:43; (i)the plant is a pepper plant, the gene or the transcript is a pepperMildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:120-122, and SEQ ID NO:123, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:49; (j) the plant is a tomato plant, the geneor the transcript is a tomato Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO:124-130, and SEQ ID NO:131, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:51 or53; (k) the plant is a wheat plant, the gene or the transcript is awheat Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:132-142, and SEQ ID NO:143, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:55, 57 or 59; (l) the plant is a grape plant,the gene or the transcript is a grape Mildew Resistance Locus O (MLO)gene or transcript, and the polynucleotide molecule is selected from thegroup consisting of SEQ ID NO:144-158, and SEQ ID NO:159 or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:61, 63,65, or 67; (m) the plant is a sorghum plant, the gene or the transcriptis a sorghum Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:164-166, and SEQ ID NO:167, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:71; or, (n) the plant is a rice plant, thegene or the transcript is a rice Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO:168-182, and SEQ ID NO:183, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:73, 75,77, or 79.

A further embodiment of the invention is directed to a plant comprisingan exogenous polynucleotide that comprises at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto a Mildew Resistance Locus O (MLO) gene or transcript of the gene,wherein the exogenous polynucleotide is not operably linked to apromoter or to a viral vector, is not integrated into the chromosomalDNA of the plant, and is not found in a non-transgenic plant; and,wherein the plant exhibits an improvement in fungal disease resistanceand/or nematode resistance that results from suppression of the MildewResistance Locus O (MLO) gene. In certain embodiments, plant furthercomprises an organosilicone compound or a component thereof. In certainembodiments, the polynucleotide is selected from the group consisting ofSEQ ID NO: 80-195, or comprises at least 18 contiguous nucleotides thatare essentially identical or essentially complementary to SEQ ID NO: 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, or 79. In some embodiments, a polynucleotidethat comprises a nucleotide sequence that is essentially identical oressentially complementary to at least 18 contiguous nucleotides of SEQID NO: 197-213 is provided. In some embodiments, the polynucleotidecomprises a nucleotide sequence that is essentially identical oressentially complementary to SEQ ID NO: 197-213. In certain embodiments:(a) the plant is a corn plant, the gene or the transcript is a cornMildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO: 160-162, and SEQ ID NO: 163, or the polynucleotide comprises atleast 18 contiguous nucleotides that are essentially identical oressentially complementary to SEQ ID NO:68-69; (b) the plant is a soybeanplant, the gene or the transcript is a soybean Mildew Resistance Locus O(MLO) gene or transcript, and the polynucleotide molecule is selectedfrom the group consisting of SEQ ID NO: 112-118, and SEQ ID NO: 119, orthe polynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:45 or47, (c) the plant is a cotton plant, the gene or the transcript is acotton Mildew Resistance Locus O (MLO) gene or transcript, and or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to a cotton gene ortranscript that encodes SEQ ID NO:4; (d) the plant is a barley plant,the gene or the transcript is a barley Mildew Resistance Locus O (MLO)gene or transcript, and the polynucleotide molecule is selected from thegroup consisting of SEQ ID NO:80-83, 184, 192, and SEQ ID NO: 197-213,or the polynucleotide comprises at least 18 contiguous nucleotides thatare essentially identical or essentially complementary to SEQ ID NO:29;(e) the plant is a cucumber plant, the gene or the transcript is acucumber Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:84-98, and SEQ ID NO:99, or the polynucleotide comprises at least 18contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:31, 33, 35, or 37; (f) the plant is a lettuceplant, the gene or the transcript is a lettuce Mildew Resistance Locus O(MLO) gene or transcript, and the polynucleotide molecule is selectedfrom the group consisting of SEQ ID NO:100-102, and SEQ ID NO:103, orthe polynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:39; (g)the plant is a pea plant, the gene or the transcript is a pea MildewResistance Locus O (MLO) gene or transcript, and the polynucleotidemolecule is selected from the group consisting of SEQ ID NO:104-106, andSEQ ID NO:107, or the polynucleotide comprises at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto SEQ ID NO:41; (h) the plant is a Medicago plant, the gene or thetranscript is a Medicago Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO:108-110, and SEQ ID NO:111, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:43; (i)the plant is a pepper plant, the gene or the transcript is a pepperMildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:120-122, and SEQ ID NO:123, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:49; (j) the plant is a tomato plant, the geneor the transcript is a tomato Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO:124-130, and SEQ ID NO:131, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:51 or53; (k) the plant is a wheat plant, the gene or the transcript is awheat Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:132-142, and SEQ ID NO:143, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:55, 57 or 59; (l) the plant is a grape plant,the gene or the transcript is a grape Mildew Resistance Locus O (MLO)gene or transcript, and the polynucleotide molecule is selected from thegroup consisting of SEQ ID NO:144-158, and SEQ ID NO:159 or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:61, 63,65, or 67; (m) the plant is a sorghum plant, the gene or the transcriptis a sorghum Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:164-166, and SEQ ID NO:167, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:71; or, (n) the plant is a rice plant, thegene or the transcript is a rice Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO:168-182, and SEQ ID NO:183, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:73, 75,77, or 79.

An additional embodiment of the invention is directed to a plant partcomprising an exogenous polynucleotide that comprises at least 18contiguous nucleotides that are essentially identical or essentiallycomplementary to a Mildew Resistance Locus O (MLO) gene or transcript ofthe gene, wherein the exogenous polynucleotide is not operably linked toa promoter or to a viral vector and is not found in a non-transgenicplant; and, wherein the plant part exhibits an improvement in fungaldisease resistance and/or nematode resistance that results fromsuppression of the Mildew Resistance Locus O (MLO) gene. In certainembodiments, the polynucleotide is selected from the group consisting ofSEQ ID NO: 80-195, or wherein the polynucleotide comprises at least 18contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, or 79. In someembodiments, a polynucleotide that comprises a nucleotide sequence thatis essentially identical or essentially complementary to at least 18contiguous nucleotides of SEQ ID NO: 197-213 is provided. In someembodiments, the polynucleotide comprises a nucleotide sequence that isessentially identical or essentially complementary to SEQ ID NO:197-213. In certain embodiments: (a) the plant part is a corn plantpart, the gene or the transcript is a corn Mildew Resistance Locus O(MLO) gene or transcript, and the polynucleotide molecule is selectedfrom the group consisting of SEQ ID NO: 160-162, and SEQ ID NO: 163, orthe polynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:68-69;(b) the plant part is a soybean plant part, the gene or the transcriptis a soybean Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO: 112-118, and SEQ ID NO: 119, or the polynucleotide comprises atleast 18 contiguous nucleotides that are essentially identical oressentially complementary to SEQ ID NO:45 or 47, (c) the plant part is acotton plant part, the gene or the transcript is a cotton MildewResistance Locus O (MLO) gene or transcript, and or the polynucleotidecomprises at least 18 contiguous nucleotides that are essentiallyidentical or essentially complementary to a cotton gene or transcriptthat encodes SEQ ID NO:4; (d) the plant part is a barley plant part, thegene or the transcript is a barley Mildew Resistance Locus O (MLO) geneor transcript, and the polynucleotide molecule is selected from thegroup consisting of SEQ ID NO:80-83, 184, 192, 194, and SEQ ID NO:197-213, or the polynucleotide comprises at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto SEQ ID NO:29; (e) the plant part is a cucumber plant part, the geneor the transcript is a cucumber Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO:84-98, and SEQ ID NO:99, or the polynucleotidecomprises at least 18 contiguous nucleotides that are essentiallyidentical or essentially complementary to SEQ ID NO:31, 33, 35, or 37;(f) the plant part is a lettuce plant part, the gene or the transcriptis a lettuce Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:100-102, and SEQ ID NO:103, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:39; (g) the plant part is a pea plant part,the gene or the transcript is a pea Mildew Resistance Locus O (MLO) geneor transcript, and the polynucleotide molecule is selected from thegroup consisting of SEQ ID NO:104-106, and SEQ ID NO:107, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:41; (h)the plant part is a Medicago plant part, the gene or the transcript is aMedicago Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:108-110, and SEQ ID NO:111, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:43; (i) the plant part is a pepper plantpart, the gene or the transcript is a pepper Mildew Resistance Locus O(MLO) gene or transcript, and the polynucleotide molecule is selectedfrom the group consisting of SEQ ID NO:120-122, and SEQ ID NO:123, orthe polynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:49; (j)the plant part is a tomato plant part, the gene or the transcript is atomato Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:124-130, and SEQ ID NO:131, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:51 or 53; (k) the plant part is a wheat plantpart, the gene or the transcript is a wheat Mildew Resistance Locus O(MLO) gene or transcript, and the polynucleotide molecule is selectedfrom the group consisting of SEQ ID NO:132-142, and SEQ ID NO:143, orthe polynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:55, 57or 59; (l) the plant part is a grape plant part, the gene or thetranscript is a grape Mildew Resistance Locus O (MLO) gene ortranscript, and the polynucleotide molecule is selected from the groupconsisting of SEQ ID NO:144-158, and SEQ ID NO:159 or the polynucleotidecomprises at least 18 contiguous nucleotides that are essentiallyidentical or essentially complementary to SEQ ID NO:61, 63, 65, or 67;(m) the plant part is a sorghum plant part, the gene or the transcriptis a sorghum Mildew Resistance Locus O (MLO) gene or transcript, and thepolynucleotide molecule is selected from the group consisting of SEQ IDNO:164-166, and SEQ ID NO:167, or the polynucleotide comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to SEQ ID NO:71; or, (n) the plant part is a rice plantpart, the gene or the transcript is a rice Mildew Resistance Locus O(MLO) gene or transcript, and the polynucleotide molecule is selectedfrom the group consisting of SEQ ID NO:168-182, and SEQ ID NO:, or thepolynucleotide comprises at least 18 contiguous nucleotides that areessentially identical or essentially complementary to SEQ ID NO:73, 75,77, or 79. In certain embodiments, the plant part is a flower, meristem,ovule, stem, tuber, fruit, anther, pollen, leaf, root, or seed. Incertain embodiments, the plant part is a seed. Also provided areprocessed plant products obtained from any of the aforementioned plantparts, wherein the processed plant products exhibit an improvedattribute relative to a processed plant product of an untreated controlplant and wherein the improved attribute results from the improvedfungal disease resistance and/or nematode resistance. In certainembodiments, the processed product is a meal, a pulp, a feed, or a foodproduct. Another embodiment of the invention is directed to a plant thatexhibits an improvement in fungal disease resistance and/or nematoderesistance, wherein the plant was topically treated with a compositionthat comprises: (a) at least one polynucleotide that comprises at least18 contiguous nucleotides that are essentially identical or essentiallycomplementary to a Mildew Resistance Locus O (MLO) gene or to atranscript of the gene; and (b) a transfer agent; and, wherein the plantexhibits an improvement in fungal disease resistance and/or nematoderesistance that results from suppression of the Mildew Resistance LocusO (MLO) gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a bootstrapped phylogenetic tree of MLO proteins

FIG. 2 presents graphs showing % disease area and leaf lengthmeasurements in untreated barley plants (NT) and barley plants treatedwith various liquid formulations as described in Table 6.

FIG. 3 presents a graph showing disease control measurements (percentageleaf area infected) in untreated barley plants (NT) and barley plantstreated with various liquid formulations as described in Table 12.

FIG. 4 presents a graph showing disease control measurements (percentageleaf area infected) in barley plants treated with short dsRNApolynucleotides at 6 day post-infection.

FIG. 5 presents a graph showing disease control measurements (percentageleaf area infected) in barley plants treated with short dsRNApolynucleotides at 13 day post-infection.

FIG. 6 presents a graph showing disease control measurements (percentageleaf area infected) in barley plants treated with long dsRNApolynucleotides at 6 day post-infection.

DETAILED DESCRIPTION

I. Definitions

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

Where a term is provided in the singular, the inventors also contemplateaspects of the invention described by the plural of that term.

As used herein, the terms “DNA,” “DNA molecule,” and “DNA polynucleotidemolecule” refer to a single-stranded DNA or double-stranded DNA moleculeof genomic or synthetic origin, such as, a polymer ofdeoxyribonucleotide bases or a DNA polynucleotide molecule.

As used herein, the terms “DNA sequence,” “DNA nucleotide sequence,” and“DNA polynucleotide sequence” refer to the nucleotide sequence of a DNAmolecule.

As used herein, the term “gene” refers to any portion of a nucleic acidthat provides for expression of a transcript or encodes a transcript. A“gene” thus includes, but is not limited to, a promoter region, 5′untranslated regions, transcript encoding regions that can includeintronic regions, and 3′ untranslated regions.

As used herein, the terms “RNA,” “RNA molecule,” and “RNA polynucleotidemolecule” refer to a single-stranded RNA or double-stranded RNA moleculeof genomic or synthetic origin, such as, a polymer of ribonucleotidebases that comprise single or double stranded regions.

Unless otherwise stated, nucleotide sequences in the text of thisspecification are given, when read from left to right, in the 5′ to 3′direction. The nomenclature used herein is that required by Title 37 ofthe United States Code of Federal Regulations § 1.822 and set forth inthe tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

As used herein, a “plant surface” refers to any exterior portion of aplant. Plant surfaces thus include, but are not limited to, the surfacesof flowers, stems, tubers, fruit, anthers, pollen, leaves, roots, orseeds. A plant surface can be on a portion of a plant that is attachedto other portions of a plant or on a portion of a plant that is detachedfrom the plant.

As used herein, the phrase “polynucleotide is not operably linked to apromoter” refers to a polynucleotide that is not covalently linked to apolynucleotide promoter sequence that is specifically recognized byeither a DNA dependent RNA polymerase II protein or by a viral RNAdependent RNA polymerase in such a manner that the polynucleotide willbe transcribed by the DNA dependent RNA polymerase II protein or viralRNA dependent RNA polymerase. A polynucleotide that is not operablylinked to a promoter can be transcribed by a plant RNA dependent RNApolymerase.

As used herein, any polynucleotide sequences of SEQ ID NO: 80-195,though displayed in the sequence listing in the form of ssDNA, encompassall other polynucleotide forms such as dsDNA equivalents, ssDNAequivalents, ssRNA equivalents, ssRNA complements, dsRNA, and ssDNAcomplements.

As used herein, any polynucleotide sequences of SEQ ID NO: 197-213,though displayed in the sequence listing in the form of one strand of adsRNA molecule, encompass all other polynucleotide forms such as dsDNAequivalents, ssDNA equivalents, ssRNA equivalents, ssRNA complements,dsRNA, and ssDNA complements.

As used herein, a first nucleic-acid sequence is “operably” connected or“linked” with a second nucleic acid sequence when the first nucleic acidsequence is placed in a functional relationship with the second nucleicacid sequence. For instance, a promoter is operably linked to an RNAand/or protein-coding sequence if the promoter provides fortranscription or expression of the RNA or coding sequence. Generally,operably linked DNA sequences are contiguous and, where necessary tojoin two protein-coding regions, are in the same reading frame.

As used herein, the phrase “organosilicone preparation” refers to aliquid comprising one or more organosilicone compounds, wherein theliquid or components contained therein, when combined with apolynucleotide in a composition that is topically applied to a targetplant surface, enable the polynucleotide to enter a plant cell. Examplesof organosilicone preparations include, but are not limited to,preparations marketed under the trade names “Silwet®” or “BREAK-THRU®”and preparations provided in Table 1. In certain embodiments, anorganosilicone preparation can enable a polynucleotide to enter a plantcell in a manner permitting a polynucleotide suppression of target geneexpression in the plant cell.

As used herein, the phrase “provides for an improvement in fungaldisease resistance and/or nematode resistance” refers to any measurableincrease in a plant's resistance to fungal- and/or nematode-induceddamage. In certain embodiments, an improvement in fungal diseaseresistance and/or nematode resistance in a plant or plant part can bedetermined in a comparison to a control plant or plant part that has notbeen treated with a composition comprising a polynucleotide and atransfer agent. When used in this context, a control plant is a plantthat has not undergone treatment with polynucleotide and a transferagent. Such control plants would include, but are not limited to,untreated plants or mock treated plants.

As used herein, the phrase “provides for a reduction”, when used in thecontext of a transcript or a protein in a plant or plant part, refers toany measurable decrease in the level of transcript or protein in a plantor plant part. In certain embodiments, a reduction of the level of atranscript or protein in a plant or plant part can be determined incomparison to a control plant or plant part that has not been treatedwith a composition comprising a polynucleotide and a transfer agent.When used in this context, a control plant or plant part is a plant orplant part that has not undergone treatment with polynucleotide and atransfer agent. Such control plants or plant parts would include, butare not limited to, untreated or mock treated plants and plant parts.

As used herein, the phrase “wherein said plant does not comprise atransgene” refers to a plant that lacks either a DNA molecule comprisinga promoter that is operably linked to an exogenous polynucleotide or arecombinant viral vector.

As used herein, the phrase “suppressing expression” or “suppression”,when used in the context of a gene, refers any measurable decrease inthe amount and/or activity of a product encoded by the gene. Thus,expression of a gene can be suppressed when there is a reduction inlevels of a transcript from the gene, a reduction in levels of a proteinencoded by the gene, a reduction in the activity of the transcript fromthe gene, a reduction in the activity of a protein encoded by the gene,any one of the preceding conditions, or any combination of the precedingconditions. In this context, the activity of a transcript includes, butis not limited to, its ability to be translated into a protein and/or toexert any RNA-mediated biologic or biochemical effect. In this context,the activity of a protein includes, but is not limited to, its abilityto exert any protein-mediated biologic or biochemical effect. In certainembodiments, a suppression of gene expression in a plant or plant partcan be determined in a comparison of gene product levels or activitiesin a treated plant to a control plant or plant part. When used in thiscontext, a control plant or plant part is a plant or plant part that hasnot undergone treatment with a composition comprising a polynucleotideand a transfer agent. Such control plants or plant parts would include,but are not limited to, untreated or mock treated plants and plantparts.

As used herein, the term “transcript” corresponds to any RNA that isproduced from a gene by the process of transcription. A transcript of agene can thus comprise a primary transcription product which can containintrons or can comprise a mature RNA that lacks introns.

As used herein, the term “liquid” refers to both homogeneous mixturessuch as solutions and non-homogeneous mixtures such as suspensions,colloids, micelles, and emulsions.

II. Overview

Provided herein are certain methods and polynucleotide compositions thatcan be applied to living plant cells/tissues to suppress expression oftarget genes and that provide improved fungal disease resistance and/ornematode resistance to a crop plant. Also provided herein are plants andplant parts exhibiting fungal disease resistance and/or nematoderesistance as well as processed products of such plants or plant parts.The compositions may be topically applied to the surface of a plant,such as to the surface of a leaf, and include a transfer agent. Aspectsof the method can be applied to various crops, for example, includingbut not limited to: i) row crop plants including, but are not limitedto, corn, barley, sorghum, soybean, cotton, canola, sugar beet, alfalfa,sugarcane, rice, and wheat; ii) vegetable plants including, but notlimited to, tomato, potato, sweet pepper, hot pepper, melon, watermelon,cucumber, eggplant, cauliflower, broccoli, lettuce, spinach, onion,peas, carrots, sweet corn, Chinese cabbage, leek, fennel, pumpkin,squash or gourd, radish, Brussels sprouts, tomatillo, garden beans, drybeans, or okra; iii) culinary plants including, but not limited to,basil, parsley, coffee, or tea; iv) fruit plants including but notlimited to apple, pear, cherry, peach, plum, apricot, banana, plantain,table grape, wine grape, citrus, avocado, mango, or berry; v) a treegrown for ornamental or commercial use, including, but not limited to, afruit or nut tree; or, vi) an ornamental plant (e. g., an ornamentalflowering plant or shrub or turf grass). The methods and compositionsprovided herein can also be applied to plants produced by a cutting,cloning, or grafting process (i. e., a plant not grown from a seed) thatinclude fruit trees and plants. Fruit trees produced by such processesinclude, but are not limited to, citrus and apple trees. Plants producedby such processes include, but are not limited to, avocados, tomatoes,eggplant, cucumber, melons, watermelons, and grapes as well as variousornamental plants.

Without being bound by a particular theory, the compositions and methodsof the present invention are believed to operate through one or more ofthe several natural cellular pathways involved in RNA-mediated genesuppression as generally described in Brodersen and Voinnet (2006),Trends Genetics, 22:268-280; Tomari and Zamore (2005) Genes & Dev.,19:517-529; Vaucheret (2006) Genes Dev., 20:759-771; Meins et al. (2005)Annu. Rev. Cell Dev. Biol., 21:297-318; and Jones-Rhoades et al. (2006)Annu. Rev. Plant Biol., 57:19-53. RNA-mediated gene suppressiongenerally involves a double-stranded RNA (dsRNA) intermediate that isformed intra-molecularly within a single RNA molecule orinter-molecularly between two RNA molecules. This longer dsRNAintermediate is processed by a ribonuclease of the RNAase III family(Dicer or Dicer-like ribonuclease) to one or more shorterdouble-stranded RNAs, one strand of which is incorporated into theRNA-induced silencing complex (“RISC”). For example, the siRNA pathwayinvolves the cleavage of a longer double-stranded RNA intermediate tosmall interfering RNAs (“siRNAs”). The size of siRNAs is believed torange from about 19 to about 25 base pairs, but the most common classesof siRNAs in plants include those containing 21 to 24 base pairs (See,Hamilton et al. (2002) EMBO J., 21:4671-4679).

Polynucleotides

As used herein, “polynucleotide” refers to a DNA or RNA moleculecontaining multiple nucleotides and generally refers both to“oligonucleotides” (a polynucleotide molecule of 18-25 nucleotides inlength) and longer polynucleotides of 26 or more nucleotides.Embodiments of this invention include compositions includingpolynucleotides having a length of 18-25 nucleotides (18-mers, 19-mers,20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), ormedium-length polynucleotides having a length of 26 or more nucleotides(polynucleotides of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about90, about 95, about 100, about 110, about 120, about 130, about 140,about 150, about 160, about 170, about 180, about 190, about 200, about210, about 220, about 230, about 240, about 250, about 260, about 270,about 280, about 290, or about 300 nucleotides), or long polynucleotideshaving a length greater than about 300 nucleotides (e. g.,polynucleotides of between about 300 to about 400 nucleotides, betweenabout 400 to about 500 nucleotides, between about 500 to about 600nucleotides, between about 600 to about 700 nucleotides, between about700 to about 800 nucleotides, between about 800 to about 900nucleotides, between about 900 to about 1000 nucleotides, between about300 to about 500 nucleotides, between about 300 to about 600nucleotides, between about 300 to about 700 nucleotides, between about300 to about 800 nucleotides, between about 300 to about 900nucleotides, or about 1000 nucleotides in length, or even greater thanabout 1000 nucleotides in length, for example up to the entire length ofa target gene including coding or non-coding or both coding andnon-coding portions of the target gene). Where a polynucleotide isdouble-stranded, its length can be similarly described in terms of basepairs.

Polynucleotide compositions used in the various embodiments of thisinvention include compositions including: RNA or DNA or RNA/DNA hybridsor chemically modified polynucleotides or a mixture thereof. In certainembodiments, the polynucleotide may be a combination of ribonucleotidesand deoxyribonucleotides, for example, synthetic polynucleotidesconsisting mainly of ribonucleotides but with one or more terminaldeoxyribonucleotides or synthetic polynucleotides consisting mainly ofdeoxyribonucleotides but with one or more terminaldideoxyribonucleotides. In certain embodiments, the polynucleotideincludes non-canonical nucleotides such as inosine, thiouridine, orpseudouridine. In certain embodiments, the polynucleotide includeschemically modified nucleotides. Examples of chemically modifiedpolynucleotides are well known in the art; see, for example, U.S. PatentPublication 2011/0171287, U.S. Patent Publication 2011/0171176, U.S.Patent Publication 2011/0152353, U.S. Patent Publication 2011/0152346,and U.S. Patent Publication 2011/0160082, which are herein incorporatedby reference. Illustrative examples include, but are not limited to, thenaturally occurring phosphodiester backbone of a polynucleotide whichcan be partially or completely modified with phosphorothioate,phosphorodithioate, or methylphosphonate internucleotide linkagemodifications, modified nucleoside bases or modified sugars can be usedin polynucleotide synthesis, and polynucleotides can be labeled with afluorescent moiety (e. g., fluorescein or rhodamine) or other label (e.g., biotin).

Polynucleotides can be single- or double-stranded RNA, single- ordouble-stranded DNA, double-stranded DNA/RNA hybrids, and modifiedanalogues thereof. In certain embodiments of the invention, thepolynucleotides that provide single-stranded RNA in the plant cell maybe: (a) a single-stranded RNA molecule (ssRNA), (b) a single-strandedRNA molecule that self-hybridizes to form a double-stranded RNAmolecule, (c) a double-stranded RNA molecule (dsRNA), (d) asingle-stranded DNA molecule (ssDNA), (e) a single-stranded DNA moleculethat self-hybridizes to form a double-stranded DNA molecule, (f) asingle-stranded DNA molecule including a modified Pol III gene that istranscribed to an RNA molecule, (g) a double-stranded DNA molecule(dsDNA), (h) a double-stranded DNA molecule including a modified Pol IIIgene that is transcribed to an RNA molecule, and (i) a double-stranded,hybridized RNA/DNA molecule, or combinations thereof. In certainembodiments, these polynucleotides can comprise both ribonucleic acidresidues and deoxyribonucleic acid residues. In certain embodiments,these polynucleotides include chemically modified nucleotides ornon-canonical nucleotides. In certain embodiments of the methods, thepolynucleotides include double-stranded DNA formed by intramolecularhybridization, double-stranded DNA formed by intermolecularhybridization, double-stranded RNA formed by intramolecularhybridization, or double-stranded RNA formed by intermolecularhybridization. In certain embodiments where the polynucleotide is adsRNA, the anti-sense strand will comprise at least 18 nucleotides thatare essentially complementary to the target gene. In certain embodimentsthe polynucleotides include single-stranded DNA or single-stranded RNAthat self-hybridizes to form a hairpin structure having an at leastpartially double-stranded structure including at least one segment thatwill hybridize to RNA transcribed from the gene targeted forsuppression. Not intending to be bound by any mechanism, it is believedthat such polynucleotides are or will produce single-stranded RNA withat least one segment that will hybridize to RNA transcribed from thegene targeted for suppression. In certain embodiments, thepolynucleotides can be operably linked to a promoter—generally apromoter functional in a plant, for example, a pol II promoter, a polIII promoter, a pol IV promoter, or a pol V promoter.

The polynucleotide molecules of the present invention are designed tomodulate expression by inducing regulation or suppression of anendogenous gene in a plant and are designed to have a nucleotidesequence essentially identical or essentially complementary to thenucleotide sequence of an endogenous gene of a plant or to the sequenceof RNA transcribed from an endogenous gene of a plant, which can becoding sequence or non-coding sequence. These effective polynucleotidemolecules that modulate expression are referred to herein as “a trigger,or triggers”. By “essentially identical” or “essentially complementary”it is meant that the trigger polynucleotides (or at least one strand ofa double-stranded polynucleotide) have sufficient identity orcomplementarity to the endogenous gene or to the RNA transcribed fromthe endogenous gene (e.g. the transcript) to suppress expression of theendogenous gene (e.g., to effect a reduction in levels or activity ofthe gene transcript and/or encoded protein). Polynucleotides of themethods and compositions provided herein need not have 100 percentidentity or complementarity to the endogenous gene or to the RNAtranscribed from the endogenous gene (i.e. the transcript) to suppressexpression of the endogenous gene (i.e. to effect a reduction in levelsor activity of the gene transcript or encoded protein). Thus, in certainembodiments, the polynucleotide or a portion thereof is designed to beessentially identical to, or essentially complementary to, a sequence ofat least 18 or 19 contiguous nucleotides in either the target gene ormessenger RNA transcribed from the target gene (e.g. the transcript). Incertain embodiments, an “essentially identical” polynucleotide has 100percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity whencompared to the sequence of 18 or more contiguous nucleotides in eitherthe endogenous target gene or to an RNA transcribed from the target gene(e.g. the transcript). In certain embodiments, an “essentiallycomplementary” polynucleotide has 100 percent sequence complementarityor at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, or 99 percent sequence complementarity when compared to thesequence of 18 or more contiguous nucleotides in either the target geneor RNA transcribed from the target gene.

In certain embodiments, polynucleotides used in the methods andcompositions provided herein can be essentially identical or essentiallycomplementary to any of: i) conserved regions of Mildew Resistance LocusO (MLO) genes of both monocot and dicot plants; ii) conserved regions ofMildew Resistance Locus O (MLO) genes of monocot plants; or iii)conserved regions of Mildew Resistance Locus O (MLO) genes of dicotplants. Such polynucleotides that are essentially identical oressentially complementary to such conserved regions can be used toimprove fungal disease resistance and/or nematode disease resistance bysuppressing expression of Mildew Resistance Locus O (MLO) genes in anyof: i) both dicot and monocot plants, including, but not limited to,corn, barley, wheat, sorghum, rice, cucumber, pea, Medicago sp.,soybean, pepper, tomato, and grape; ii) monocot plants, including, butnot limited to, corn, barley, wheat, sorghum, and rice, and; or iii)dicot plants, including, but not limited to, cucumber, pea, Medicagosp., soybean, pepper, tomato, and grape.

Polynucleotides containing mismatches to the target gene or transcriptcan thus be used in certain embodiments of the compositions and methodsprovided herein. In certain embodiments, a polynucleotide can compriseat least 19 contiguous nucleotides that are essentially identical oressentially complementary to said gene or said transcript or comprisesat least 19 contiguous nucleotides that are essentially identical oressentially complementary to the target gene or target gene transcript.In certain embodiments, a polynucleotide of 19 continuous nucleotidesthat is essentially identical or essentially complementary to theendogenous target gene or to RNA transcribed from the target gene (e.g.the transcript) can have 1 or 2 mismatches to the target gene ortranscript. In certain embodiments, a polynucleotide of 20 or morenucleotides that contains a contiguous 19 nucleotide span of identity orcomplementarity to the endogenous target gene or to an RNA transcribedfrom the target gene can have 1 or 2 mismatches to the target gene ortranscript. In certain embodiments, a polynucleotide of 21 continuousnucleotides that is essentially identical or essentially complementaryto the endogenous target gene or to RNA transcribed from the target gene(e.g. the transcript) can have 1, 2, or 3 mismatches to the target geneor transcript. In certain embodiments, a polynucleotide of 22 or morenucleotides that contains a contiguous 21 nucleotide span of identity orcomplementarity to the endogenous target gene or to an RNA transcribedfrom the target gene can have 1, 2, or 3 mismatches to the target geneor transcript. In designing polynucleotides with mismatches to anendogenous target gene or to an RNA transcribed from the target gene,mismatches of certain types and at certain positions that are morelikely to be tolerated can be used. In certain embodiments, mismatchesformed between adenine and cytosine or guanosine and uracil residues areused as described by Du et al. Nucleic Acids Research, 2005, Vol. 33,No. 5 1671-1677. In certain embodiments, mismatches in 19 base pairoverlap regions can be at the low tolerance positions 5, 7, 8 or 11(from the 5′ end of a 19 nucleotide target) with well toleratednucleotide mismatch residues, at medium tolerance positions 3, 4, and12-17, and/or at the high tolerance nucleotide positions at either endof the region of complementarity (i.e. positions 1, 2, 18, and 19) asdescribed by Du et al. Nucleic Acids Research, 2005, Vol. 33, No. 51671-1677. It is further anticipated that tolerated mismatches can beempirically determined in assays where the polynucleotide is applied tothe plants via the methods provided herein and the treated plantsassayed for suppression of Mildew Resistance Locus O (MLO) expression orappearance of fungal disease resistance and/or nematode resistance.

In certain embodiments, polynucleotide molecules are designed to have100 percent sequence identity with or complementarity to one allele orone family member of a given target gene coding or non-coding sequenceof a MLO target gene. In other embodiments, the polynucleotide moleculesare designed to have 100 percent sequence identity with orcomplementarity to multiple alleles or family members of a given MildewResistance Locus O (MLO) target gene. In certain embodiments, thepolynucleotide can thus comprises at least 18 contiguous nucleotidesthat are identical or complementary to SEQ ID NO: 4, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, or 79. In certain embodiments, the polynucleotide comprises atleast 18 contiguous nucleotides that are essentially identical oressentially complementary to SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, or79.

In certain embodiments, polynucleotide compositions and methods providedherein typically effect regulation or modulation (e. g., suppression) ofgene expression during a period during the life of the treated plant ofat least 1 week or longer and typically in systemic fashion. Forinstance, within days of treating a plant leaf with a polynucleotidecomposition of this invention, primary and transitive siRNAs can bedetected in other leaves lateral to and above the treated leaf and inapical tissue. In certain embodiments, methods of systemicallysuppressing expression of a gene in a plant, the methods comprisingtreating said plant with a composition comprising at least onepolynucleotide and a transfer agent, wherein said polynucleotidecomprises at least 18 or at least 19 contiguous nucleotides that areessentially identical or essentially complementary to a gene or atranscript encoding a Mildew Resistance Locus O (MLO) gene of the plantare provided, whereby expression of the gene in said plant or progenythereof is systemically suppressed in comparison to a control plant thathas not been treated with the composition.

Compositions used to suppress a target gene can comprise one or morepolynucleotides that are essentially identical or essentiallycomplementary to multiple genes, or to multiple segments of one or moregenes. In certain embodiments, compositions used to suppress a targetgene can comprise one or more polynucleotides that are essentiallyidentical or essentially complementary to multiple consecutive segmentsof a target gene, multiple non-consecutive segments of a target gene,multiple alleles of a target gene, or multiple target genes from one ormore species.

In certain embodiments, the polynucleotide includes two or more copiesof a nucleotide sequence (of 18 or more nucleotides) where the copiesare arranged in tandem fashion. In another embodiment, thepolynucleotide includes two or more copies of a nucleotide sequence (of18 or more nucleotides) where the copies are arranged in inverted repeatfashion (forming an at least partially self-complementary strand). Thepolynucleotide can include both tandem and inverted-repeat copies.Whether arranged in tandem or inverted repeat fashion, each copy can bedirectly contiguous to the next, or pairs of copies can be separated byan optional spacer of one or more nucleotides. The optional spacer canbe unrelated sequence (i. e., not essentially identical to oressentially complementary to the copies, nor essentially identical to,or essentially complementary to, a sequence of 18 or more contiguousnucleotides of the endogenous target gene or RNA transcribed from theendogenous target gene). Alternatively the optional spacer can includesequence that is complementary to a segment of the endogenous targetgene adjacent to the segment that is targeted by the copies. In certainembodiments, the polynucleotide includes two copies of a nucleotidesequence of between about 20 to about 30 nucleotides, where the twocopies are separated by a spacer no longer than the length of thenucleotide sequence.

Tiling

Polynucleotide trigger molecules can be identified by “tiling” genetargets in random length fragments, e.g., 200-300 polynucleotides inlength, with partially overlapping regions, e.g., 25 or so nucleotideoverlapping regions along the length of the target gene. Multiple genetarget sequences can be aligned and polynucleotide sequence regions withhomology in common are identified as potential trigger molecules formultiple targets. Multiple target sequences can be aligned and sequenceregions with poor homology are identified as potential trigger moleculesfor selectively distinguishing targets. To selectively suppress a singlegene, trigger sequences may be chosen from regions that are unique tothe target gene either from the transcribed region or the non-codingregions, e.g., promoter regions, 3′ untranslated regions, introns andthe like.

Polynucleotides fragments are designed along the length of the fulllength coding and untranslated regions of a MLO gene or family member ascontiguous overlapping fragments of 200-300 polynucleotides in length orfragment lengths representing a percentage of the target gene. Thesefragments are applied topically (as sense or anti-sense ssDNA or ssRNA,dsRNA, or dsDNA) to determine the relative effectiveness in providingthe yield/quality phenotype. Fragments providing the desired activitymay be further subdivided into 50-60 polynucleotide fragments which areevaluated for providing the yield/quality phenotype. The 50-60 basefragments with the desired activity may then be further subdivided into19-30 base fragments which are evaluated for providing the yield/qualityphenotype. Once relative effectiveness is determined, the fragments areutilized singly, or in combination in one or more pools to determineeffective trigger composition or mixture of trigger polynucleotides forproviding the yield/quality phenotype.

Coding and/or non-coding sequences of gene families in the crop ofinterest are aligned and 200-300 polynucleotide fragments from the leasthomologous regions amongst the aligned sequences are evaluated usingtopically applied polynucleotides (as sense or anti-sense ssDNA orssRNA, dsRNA, or dsDNA) to determine their relative effectiveness inproviding the yield/quality phenotype. The effective segments arefurther subdivided into 50-60 polynucleotide fragments, prioritized byleast homology, and reevaluated using topically applied polynucleotides.The effective 50-60 polynucleotide fragments are subdivided into 19-30polynucleotide fragments, prioritized by least homology, and againevaluated for induction of the yield/quality phenotype. Once relativeeffectiveness is determined, the fragments are utilized singly, or againevaluated in combination with one or more other fragments to determinethe trigger composition or mixture of trigger polynucleotides forproviding the yield/quality phenotype.

Coding and/or non-coding sequences of gene families in the crop ofinterest are aligned and 200-300 polynucleotide fragments from the mosthomologous regions amongst the aligned sequences are evaluated usingtopically applied polynucleotides (as sense or anti-sense ssDNA orssRNA, dsRNA, or dsDNA) to determine their relative effectiveness ininducing the yield/quality phenotype. The effective segments aresubdivided into 50-60 polynucleotide fragments, prioritized by mosthomology, and reevaluated using topically applied polynucleotides. Theeffective 50-60 polynucleotide fragments are subdivided into 19-30polynucleotide fragments, prioritized by most homology, and againevaluated for induction of the yield/quality phenotype. Once relativeeffectiveness is determined, the fragments may be utilized singly, or incombination with one or more other fragments to determine the triggercomposition or mixture of trigger polynucleotides for providing theyield/quality phenotype.

Also, provided herein are methods for identifying a preferredpolynucleotide for improving fungal disease and/or nematode resistancein a plant. Populations of candidate polynucleotides that areessentially identical or essentially complementary to a MLO gene ortranscript of the gene can be generated by a variety of approaches,including but not limited to, any of the tiling, least homology, or mosthomology approaches provided herein. Such populations of polynucleotidescan also be generated or obtained from any of the polynucleotides orgenes provided herewith in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 42,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,or 79. Such populations of polynucleotides can also be generated orobtained from any genes that are orthologous to the genes providedherewith in SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 42, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, or 79. Suchpopulations of polynucleotides can also be generated or obtained fromany genes that encode proteins that are orthologous to a protein ofTable 2 or 3 (SEQ ID NO:1-27, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78). Suchpolynucleotides can be topically applied to a surface of plants in acomposition comprising at least one polynucleotide from said populationand a transfer agent to obtain treated plants. Treated plants thatexhibit suppression of the MLO gene and/or exhibit an improvement fungaldisease and/or nematode resistance are identified, thus identifying apreferred polynucleotide that improves improving fungal disease and/ornematode resistance in a plant. Suppression of the gene can bedetermined by any assay for the levels and/or activity of a gene product(i.e., transcript or protein). Suitable assays for transcripts include,but are not limited to, semi-quantitative or quantitative reversetranscriptase PCR® (qRT-PCR) assays. Suitable assays for proteinsinclude, but are not limited to, semi-quantitative or quantitaiveimmunoassays, biochemical activity assays, or biological activityassays. In certain embodiments, the polynucleotides can be appliedalone. In other embodiments, the polynucleotides can be applied in poolsof multiple polynucleotides. When a pool of polynucleotides provides forsuppression of the MLO gene and/or an improvement in fungal diseaseresistance and/or nematode disease resistance are identified, the poolcan be de-replicated and retested as necessary or desired to identifyone or more preferred polynucleotide(s) that improves fungal diseaseresistance and/or nematode disease resistance in a plant.

Methods of making polynucleotides are well known in the art. Suchmethods of making polynucleotides can include in vivo biosynthesis, invitro enzymatic synthesis, or chemical synthesis. In certainembodiments, RNA molecules can be made by either in vivo or in vitrosynthesis from DNA templates where a suitable promoter is operablylinked to the polynucleotide and a suitable DNA-dependent RNA polymeraseis provided. DNA-dependent RNA polymerases include, but are not limitedto, E. coli or other bacterial RNA polymerases as well as thebacteriophage RNA polymerases such as the T7, T3, and SP6 RNApolymerases. Commercial preparation of polynucleotides often providestwo deoxyribonucleotides on the 3′ end of the sense strand. Longpolynucleotide molecules can be synthesized from commercially availablekits, for example, kits from Applied Biosystems/Ambion (Austin, Tex.)have DNA ligated on the 5′ end that encodes a bacteriophage T7polymerase promoter that makes RNA strands that can be assembled into adsRNA. Alternatively, dsRNA molecules can be produced from expressioncassettes in bacterial cells that have regulated or deficient RNase IIIenzyme activity. Long polynucleotide molecules can also be assembledfrom multiple RNA or DNA fragments. In some embodiments designparameters such as Reynolds score (Reynolds et al. Nature Biotechnology22, 326-330 (2004) and Tuschl rules (Pei and Tuschl, Nature Methods3(9): 670-676, 2006) are known in the art and are used in selectingpolynucleotide sequences effective in gene silencing. In someembodiments random design or empirical selection of polynucleotidesequences is used in selecting polynucleotide sequences effective ingene silencing. In some embodiments, the sequence of a polynucleotide isscreened against the genomic DNA of the intended plant to minimizeunintentional silencing of other genes.

While there is no upper limit on the concentrations and dosages ofpolynucleotide molecules that can be useful in the methods andcompositions provided herein, lower effective concentrations and dosageswill generally be sought for efficiency. The concentrations can beadjusted in consideration of the volume of spray or treatment applied toplant leaves or other plant part surfaces, such as flower petals, stems,tubers, fruit, anthers, pollen, leaves, roots, or seeds. In oneembodiment, a useful treatment for herbaceous plants using 25-merpolynucleotide molecules is about 1 nanomole (nmol) of polynucleotidemolecules per plant, for example, from about 0.05 to 1 nmolpolynucleotides per plant. Other embodiments for herbaceous plantsinclude useful ranges of about 0.05 to about 100 nmol, or about 0.1 toabout 20 nmol, or about 1 nmol to about 10 nmol of polynucleotides perplant. In certain embodiments, about 40 to about 50 nmol of a ssDNApolynucleotide are applied. In certain embodiments, about 0.5 nmol toabout 2 nmol of a dsRNA is applied. In certain embodiments, acomposition containing about 0.5 to about 2.0 mg/mL, or about 0.14 mg/mLof dsRNA or ssDNA (21-mer) is applied. In certain embodiments, acomposition of about 0.5 to about 1.5 mg/mL of a long dsRNApolynucleotide (i.e., about 50 to about 200 or more nucleotides) isapplied. In certain embodiments, about 1 nmol to about 5 nmol of a dsRNAis applied to a plant. In certain embodiments, the polynucleotidecomposition as topically applied to the plant contains the at least onepolynucleotide at a concentration of about 0.01 to about 10 milligramsper milliliter, or about 0.05 to about 2 milligrams per milliliter, orabout 0.1 to about 2 milligrams per milliliter. In certain embodiments,a composition of about 0.5 to about 1.5 mg/mL of a long dsRNApolynucleotide (i.e. about 50 to about 200 or more nucleotides) isapplied. Very large plants, trees, or vines may require correspondinglylarger amounts of polynucleotides. When using long dsRNA molecules thatcan be processed into multiple polynucleotides, lower concentrations canbe used. To illustrate embodiments of the invention, the factor 1×, whenapplied to polynucleotide molecules is arbitrarily used to denote atreatment of 0.8 nmol of polynucleotide molecule per plant; 10×, 8 nmolof polynucleotide molecule per plant; and 100×, 80 nmol ofpolynucleotide molecule per plant.

The polynucleotide compositions of this invention are useful incompositions, such as liquids that comprise polynucleotide molecules,alone or in combination with other components either in the same liquidor in separately applied liquids that provide a transfer agent. As usedherein, a transfer agent is an agent that, when combined with apolynucleotide in a composition that is topically applied to a targetplant surface, enables the polynucleotide to enter a plant cell. Incertain embodiments, a transfer agent is an agent that conditions thesurface of plant tissue, e. g., seeds, leaves, stems, roots, flowers, orfruits, to permeation by the polynucleotide molecules into plant cells.The transfer of polynucleotides into plant cells can be facilitated bythe prior or contemporaneous application of apolynucleotide-transferring agent to the plant tissue. In someembodiments the transferring agent is applied subsequent to theapplication of the polynucleotide composition. The polynucleotidetransfer agent enables a pathway for polynucleotides through cuticle waxbarriers, stomata and/or cell wall or membrane barriers into plantcells. Suitable transfer agents to facilitate transfer of thepolynucleotide into a plant cell include agents that increasepermeability of the exterior of the plant or that increase permeabilityof plant cells to polynucleotides. Such agents to facilitate transfer ofthe composition into a plant cell include a chemical agent, or aphysical agent, or combinations thereof. Chemical agents forconditioning or transfer include (a) surfactants, (b) an organic solventor an aqueous solution or aqueous mixtures of organic solvents, (c)oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, orcombinations thereof. Embodiments of the method can optionally includean incubation step, a neutralization step (e.g., to neutralize an acid,base, or oxidizing agent, or to inactivate an enzyme), a rinsing step,or combinations thereof. Embodiments of agents or treatments forconditioning of a plant to permeation by polynucleotides includeemulsions, reverse emulsions, liposomes, and other micellar-likecompositions. Embodiments of agents or treatments for conditioning of aplant to permeation by polynucleotides include counter-ions or othermolecules that are known to associate with nucleic acid molecules, e.g., inorganic ammonium ions, alkyl ammonium ions, lithium ions,polyamines such as spermine, spermidine, or putrescine, and othercations. Organic solvents useful in conditioning a plant to permeationby polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine,hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol,other solvents miscible with water or that will dissolvephosphonucleotides in non-aqueous systems (such as is used in syntheticreactions). Naturally derived or synthetic oils with or withoutsurfactants or emulsifiers can be used, e. g., plant-sourced oils, cropoils (such as those listed in the 9^(th) Compendium of HerbicideAdjuvants, publicly available on the worldwide web (internet) atherbicide.adjuvants.com can be used, e. g., paraffinic oils, polyolfatty acid esters, or oils with short-chain molecules modified withamides or polyamines such as polyethyleneimine or N-pyrrolidine.Transfer agents include, but are not limited to, organosiliconepreparations.

In certain embodiments, an organosilicone preparation that iscommercially available as Silwet® L-77 surfactant having CAS Number27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, and currentlyavailable from Momentive Performance Materials, Albany, N.Y. can be usedto prepare a polynucleotide composition. In certain embodiments where aSilwet L-77 organosilicone preparation is used as a pre-spray treatmentof plant leaves or other plant surfaces, freshly made concentrations inthe range of about 0.015 to about 2 percent by weight (wt percent) (e.g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05,0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious inpreparing a leaf or other plant surface for transfer of polynucleotidemolecules into plant cells from a topical application on the surface. Incertain embodiments of the methods and compositions provided herein, acomposition that comprises a polynucleotide molecule and anorganosilicone preparation comprising Silwet L-77 in the range of about0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01,0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065,0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,2.1, 2.2, 2.3, 2.5 wt percent) is used or provided. In certainembodiments of the methods and compositions provided herein, acomposition that comprises a polynucleotide molecule and anorganosilicone preparation comprising Silwet L-77 in the range of about0.3 to about 1 percent by weight (wt percent) or about 0.5 to about 1%.by weight (wt percent) is used or provided.

In certain embodiments, any of the commercially available organosiliconepreparations provided in the following Table 1 can be used as transferagents in a polynucleotide composition. In certain embodiments where anorganosilicone preparation of Table 1 is used as a pre-spray treatmentof plant leaves or other surfaces, freshly made concentrations in therange of about 0.015 to about 2 percent by weight (wt percent) (e. g.,about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055,0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious in preparing aleaf or other plant surface for transfer of polynucleotide moleculesinto plant cells from a topical application on the surface. In certainembodiments of the methods and compositions provided herein, acomposition that comprises a polynucleotide molecule and anorganosilicone preparation of Table 1 in the range of about 0.015 toabout 2 percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02,0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075,0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.5 wt percent) is used or provided.

TABLE 1 Examples of organosilicone preparations Name CAS numberManufacturer^(1,2) BREAK-THRU ® S 321 na Evonik Industries AGBREAK-THRU ® S 200 67674-67-3 Evonik Industries AG BREAK-THRU ® OE 44168937-55-3 Evonik Industries AG BREAK-THRU ® S 278 27306-78-1 EvonikGoldschmidt BREAK-THRU ® S 243 na Evonik Industries AG Silwet ® L-7727306-78-1 Momentive Performance Materials Silwet ® HS 429 na MomentivePerformance Materials Silwet ® HS 312 na Momentive Performance MaterialsBREAK-THRU ® S 233 134180-76-0 Evonik Industries AG Silwet ® HS 508Momentive Performance Materials Silwet ® HS 604 Momentive PerformanceMaterials ¹Evonik Industries AG, Essen, Germany ²Momentive PerformanceMaterials, Albany, New York

Organosilicone preparations used in the methods and compositionsprovided herein can comprise one or more effective organosiliconecompounds. As used herein, the phrase “effective organosiliconecompound” is used to describe any organosilicone compound that is foundin an organosilicone preparation that enables a polynucleotide to entera plant cell. In certain embodiments, an effective organosiliconecompound can enable a polynucleotide to enter a plant cell in a mannerpermitting a polynucleotide mediated suppression of a target geneexpression in the plant cell. In general, effective organosiliconecompounds include, but are not limited to, compounds that can comprise:i) a trisiloxane head group that is covalently linked to, ii) an alkyllinker including, but not limited to, an n-propyl linker, that iscovalently linked to, iii) a poly glycol chain, that is covalentlylinked to, iv) a terminal group. Trisiloxane head groups of sucheffective organosilicone compounds include, but are not limited to,heptamethyltrisiloxane. Alkyl linkers can include, but are not limitedto, an n-propyl linker. Poly glycol chains include, but are not limitedto, polyethylene glycol or polypropylene glycol. Poly glycol chains cancomprise a mixture that provides an average chain length “n” of about“7.5”. In certain embodiments, the average chain length “n” can varyfrom about 5 to about 14. Terminal groups can include, but are notlimited to, alkyl groups such as a methyl group. Effectiveorganosilicone compounds are believed to include, but are not limitedto, trisiloxane ethoxylate surfactants or polyalkylene oxide modifiedheptamethyl trisiloxane.

One organosilicone compound believed to be ineffective comprises theformula:

In certain embodiments, an organosilicone preparation that comprises anorganosilicone compound comprising a trisiloxane head group is used inthe methods and compositions provided herein. In certain embodiments, anorganosilicone preparation that comprises an organosilicone compoundcomprising a heptamethyltrisiloxane head group is used in the methodsand compositions provided herein. In certain embodiments, anorganosilicone composition that comprises Compound I is used in themethods and compositions provided herein. In certain embodiments, anorganosilicone composition that comprises Compound I is used in themethods and compositions provided herein. In certain embodiments of themethods and compositions provided herein, a composition that comprises apolynucleotide molecule and one or more effective organosiliconecompound in the range of about 0.015 to about 2 percent by weight (wtpercent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04,0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) is used orprovided.

In certain embodiments, the polynucleotide compositions that comprise anorganosilicone preparation can comprise a salt such as ammoniumchloride, tetrabutylphosphonium bromide, and/or ammonium sulfate.Ammonium chloride, tetrabutylphosphonium bromide, and/or ammoniumsulfate can be provided in the polynucleotide composition at aconcentration of about 0.5% to about 5% (w/v). An ammonium chloride,tetrabutylphosphonium bromide, and/or ammonium sulfate concentration ofabout 1% to about 3%, or about 2% (w/v) can also be used in thepolynucleotide compositions that comprise an organosilicone preparation.In certain embodiments, the polynucleotide compositions can comprise anammonium salt at a concentration greater or equal to 300 millimolar. Incertain embodiments, the polynucleotide compositions that comprise anorganosilicone preparation can comprise ammonium sulfate atconcentrations from about 80 to about 1200 mM or about 150 mM to about600 mM.

In certain embodiments, the polynucleotide compositions can alsocomprise a phosphate salt. Phosphate salts used in the compositionsinclude, but are not limited to, calcium, magnesium, potassium, orsodium phosphate salts. In certain embodiments, the polynucleotidecompositions can comprise a phosphate salt at a concentration of atleast about 5 millimolar, at least about 10 millimolar, or at leastabout 20 millimolar. In certain embodiments, the polynucleotidecompositions will comprise a phosphate salt in a range of about 1 mM toabout 25 mM or in a range of about 5 mM to about 25 mM. In certainembodiments, the polynucleotide compositions can comprise sodiumphosphate at a concentration of at least about 5 millimolar, at leastabout 10 millimolar, or at least about 20 millimolar. In certainembodiments, the polynucleotide compositions can comprise sodiumphosphate at a concentration of about 5 millimolar, about 10 millimolar,or about 20 millimolar. In certain embodiments, the polynucleotidecompositions will comprise a sodium phosphate salt in a range of about10 mM to about 160 mM or in a range of about 20 mM to about 40 mM. Incertain embodiments, the polynucleotide compositions can comprise asodium phosphate buffer at a pH of about 6.8.

In certain embodiments, other useful transfer agents or adjuvants totransfer agents that can be used in polynucleotide compositions providedherein include surfactants and/or effective molecules contained therein.Surfactants and/or effective molecules contained therein include, butare not limited to, sodium or lithium salts of fatty acids (such astallow or tallowamines or phospholipids) and organosilicone surfactants.In certain embodiments, the polynucleotide compositions that comprise atransfer agent are formulated with counter-ions or other molecules thatare known to associate with nucleic acid molecules. Illustrativeexamples include, but are not limited to, tetraalkyl ammonium ions,trialkyl ammonium ions, sulfonium ions, lithium ions, and polyaminessuch as spermine, spermidine, or putrescine. In certain embodiments, thepolynucleotide compositions are formulated with a non-polynucleotideherbicide. Non-polynucleotide herbicidal molecules include, but are notlimited to, glyphosate, auxin-like benzoic acid herbicides includingdicamba, chloramben and TBA, glufosinate, auxin-like herbicidesincluding phenoxy carboxylic acid herbicide, pyridine carboxylic acidherbicide, quinoline carboxylic acid herbicide, pyrimidine carboxylicacid herbicide, and benazolin-ethyl herbicide, sulfonylureas,imidazolinones, bromoxynil, delapon, cyclohezanedione,protoporphyrionogen oxidase inhibitors, and4-hydroxyphenyl-pyruvate-dioxygenase inhibiting herbicides.

In certain embodiments, the polynucleotides used in the compositionsthat are essentially identical or essentially complementary to the MLOtarget gene or transcript will comprise the predominant nucleic acid inthe composition. Thus in certain embodiments, the polynucleotides thatare essentially identical or essentially complementary to the targetgene or transcript will comprise at least about 50%, 75%, 95%, 98%, or100% of the nucleic acids provided in the composition by either mass ormolar concentration. However, in certain embodiments, thepolynucleotides that are essentially identical or essentiallycomplementary to the target gene or transcript can comprise at leastabout 1% to about 50%, about 10% to about 50%, about 20% to about 50%,or about 30% to about 50% of the nucleic acids provided in thecomposition by either mass or molar concentration. Also provided arecompositions where the polynucleotides that are essentially identical oressentially complementary to the target gene or transcript can compriseat least about 1% to 100%, about 10% to 100%, about 20% to about 100%,about 30% to about 50%, or about 50% to a 100% of the nucleic acidsprovided in the composition by either mass or molar concentration.

Polynucleotides comprising ssDNA, dsDNA, ssRNA, dsRNA, or RNA/DNAhybrids that are essentially identical or complementary to certain planttarget genes or transcripts and that can be used in compositionscontaining transfer agents that include, but are not limited to,organosilicone preparations, to suppress those target genes whentopically applied to plants are disclosed in co-assigned U.S. patentapplication Ser. No. 13/042,856. Various polynucleotide herbicidalmolecules, compositions comprising those polynucleotide herbicidalmolecules and transfer agents that include, but are not limited to,organosilicone preparations, and methods whereby herbicidal effects areobtained by the topical application of such compositions to plants arealso disclosed in co-assigned U.S. patent application Ser. No.13/042,856, and those polynucleotide herbicidal molecules, compositions,and methods are incorporated herein by reference in their entireties.Genes encoding proteins that can provide tolerance to an herbicideand/or that are targets of a herbicide are collectively referred toherein as “herbicide target genes”. Herbicide target genes include, butare not limited to, a 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS), a glyphosate oxidoreductase (GOX), a glyphosate decarboxylase,a glyphosate-N-acetyl transferase (GAT), a dicamba monooxygenase, aphosphinothricin acetyltransferase, a 2,2-dichloropropionic aciddehalogenase, an acetohydroxyacid synthase, an acetolactate synthase, ahaloarylnitrilase, an acetyl-coenzyme A carboxylase (ACCase), adihydropteroate synthase, a phytoene desaturase (PDS), a protoporphyrinIX oxygenase (PPO), a hydroxyphenylpyruvate dioxygenase (HPPD), apara-aminobenzoate synthase, a glutamine synthase, a cellulose synthase,a beta tubulin, and a serine hydroxymethyltransferase gene. The effectsof applying certain compositions comprising polynucleotides that areessentially identical or complementary to certain herbicide target genesand transfer agents on plants containing the herbicide target genes wasshown to be potentiated or enhanced by subsequent application of anherbicide that targets the same gene as the polynucleotide inco-assigned U.S. patent application Ser. No. 13/042,856. For example,compositions comprising polynucleotides targeting the EPSPS herbicidetarget gene were potentiated by glyphosate in experiments disclosed inco-assigned U.S. patent application Ser. No. 13/042,856.

In certain embodiments of the compositions and methods disclosed herein,the composition comprising a polynucleotide and a transfer agent canthus further comprise a second polynucleotide comprising at least 19contiguous nucleotides that are essentially identical or essentiallycomplementary to a transcript to a protein that confers resistance to aherbicide. In certain embodiments, the second polynucleotide does notcomprise a polynucleotide that is essentially identical or essentiallycomplementary to a transcript encoding a protein of a target plant thatconfers resistance to said herbicidal molecule. Thus, in an exemplaryand non-limiting embodiment, the second polynucleotide could beessentially identical or essentially complementary to a transcriptencoding a protein that confers resistance to a herbicide in a weed(such as an EPSPS encoding transcript) but would not be essentiallyidentical or essentially complementary to a transcript encoding aprotein that confers resistance to that same herbicide in a crop plant.

In certain embodiments, the polynucleotide compositions that comprise atransfer agent can comprise glycerin. Glycerin can be provided in thecomposition at a concentration of about 0.1% to about 1% (w/v or v/v). Aglycerin concentration of about 0.4% to about 0.6%, or about 0.5% (w/vor v/v) can also be used in the polynucleotide compositions thatcomprise a transfer agent.

In certain embodiments, the polynucleotide compositions that comprise atransfer agent can further comprise organic solvents. Such organicsolvents include, but are not limited to, DMSO, DMF, pyridine,N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane,polypropylene glycol, other solvents miscible with water or that willdissolve phosphonucleotides in non-aqueous systems (such as is used insynthetic reactions).

In certain embodiments, the polynucleotide compositions that comprise atransfer agent can further comprise naturally derived or synthetic oilswith or without surfactants or emulsifiers. Such oils include, but arenot limited to, plant-sourced oils, crop oils (such as those listed inthe 9th Compendium of Herbicide Adjuvants, publicly available on line atwww.herbicide.adjuvants.com), paraffinic oils, polyol fatty acid esters,or oils with short-chain molecules modified with amides or polyaminessuch as polyethyleneimine or N-pyrrolidine.

In aspects of the invention, methods include one or more applications ofthe composition comprising a polynucleotide and a transfer agent or oneor more effective components contained therein. In certain embodimentsof the methods, one or more applications of a transfer agent or one ormore effective components contained therein can precede one or moreapplications of the composition comprising a polynucleotide and atransfer agent. In embodiments where a transfer agent and/or one or moreeffective molecules contained therein is used either by itself as apre-treatment or as part of a composition that includes apolynucleotide, embodiments of the polynucleotide molecules aredouble-stranded RNA polynucleotides, single-stranded RNApolynucleotides, double-stranded DNA polynucleotides, single-strandedDNA polynucleotides, chemically modified RNA or DNA polynucleotides ormixtures thereof.

Compositions and methods of the invention are useful for modulating orsuppressing the expression of an endogenous Mildew Resistance Locus O(MLO) target gene or transgenic Mildew Resistance Locus O (MLO) targetgene in a plant cell or plant. In certain embodiments of the methods andcompositions provided herein, expression of MLO target genes can besuppressed completely, partially and/or transiently to result in animprovement in fungal disease resistance and/or nematode resistance. Invarious embodiments, a Mildew Resistance Locus O (MLO) target geneincludes coding (protein-coding or translatable) sequence, non-coding(non-translatable) sequence, or both coding and non-coding sequence.Compositions of the invention can include polynucleotides designed totarget multiple Mildew Resistance Locus O (MLO) genes, or multiplesegments of one or more Mildew Resistance Locus O (MLO) genes. Thetarget gene can include multiple consecutive segments of a target MildewResistance Locus O (MLO) gene, multiple non-consecutive segments of aMildew Resistance Locus O (MLO) target gene, multiple alleles of atarget gene, or multiple Mildew Resistance Locus O (MLO) target genesfrom one or more species. Mildew Resistance Locus O (MLO) target genesinclude, but are not limited to, the endogenous Mildew Resistance LocusO (MLO) plant genes of SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, or 79.Mildew Resistance Locus O (MLO) target genes include, but are notlimited to, Mildew Resistance Locus O (MLO) plant genes that encodeproteins that are orthologous to the proteins of SEQ ID NO: 1-28. MildewResistance Locus O (MLO) target genes include, but are not limited to,Mildew Resistance Locus O (MLO) plant genes that encode the proteins ofSEQ ID NO: 1-28.

Target genes and plants containing those target genes can be obtainedfrom: i) row crop plants including, but are not limited to, corn,soybean, cotton, canola, sugar beet, alfalfa, sugarcane, rice, andwheat; ii) vegetable plants including, but not limited to, tomato,potato, sweet pepper, hot pepper, melon, watermelon, cucumber, eggplant,cauliflower, broccoli, lettuce, spinach, onion, peas, carrots, sweetcorn, Chinese cabbage, leek, fennel, pumpkin, squash or gourd, radish,Brussels sprouts, tomatillo, garden beans, dry beans, or okra; iii)culinary plants including, but not limited to, basil, parsley, coffee,or tea; iv) fruit plants including but not limited to apple, pear,cherry, peach, plum, apricot, banana, plantain, table grape, wine grape,citrus, avocado, mango, or berry; v) a tree grown for ornamental orcommercial use, including, but not limited to, a fruit or nut tree; or,vi) an ornamental plant (e. g., an ornamental flowering plant or shrubor turf grass). The methods and compositions provided herein can also beapplied to plants produced by a cutting, cloning, or grafting process(i. e., a plant not grown from a seed) include fruit trees and plantsthat include, but are not limited to, citrus, apples, avocados,tomatoes, eggplant, cucumber, melons, watermelons, and grapes as well asvarious ornamental plants. Such row crop, vegetable, culinary, fruit,tree, or ornamental plants exhibiting improvements in fungal diseaseresistance and/or nematode resistance that result from suppressingMildew Resistance Locus O (MLO) gene expression are provided herein.Such row crop, vegetable, culinary, fruit, tree, or ornamental plantparts or processed plant products exhibiting improvements in fungaldisease resistance and/or nematode resistance that result fromsuppressing Mildew Resistance Locus O (MLO) gene expression are alsoprovided herein. Such plant parts can include, but are not limited to,flowers, stems, tubers, fruit, anthers, meristems, ovules, pollen,leaves, or seeds. Such processed plant products obtained from the plantparts can include, but are not limited to, a meal, a pulp, a feed, or afood product.

An aspect of the invention provides a method for modulating orsuppressing expression of an Mildew Resistance Locus O (MLO) gene in aplant including (a) conditioning of a plant to permeation bypolynucleotides and (b) treatment of the plant with the polynucleotidemolecules, wherein the polynucleotide molecules include at least onesegment of 18 or more contiguous nucleotides cloned from or otherwiseidentified from the Mildew Resistance Locus O (MLO) target gene ineither anti-sense or sense orientation, whereby the polynucleotidemolecules permeate the interior of the plant and induce modulation ofthe target gene. The conditioning and polynucleotide application can beperformed separately or in a single step. When the conditioning andpolynucleotide application are performed in separate steps, theconditioning can precede or can follow the polynucleotide applicationwithin minutes, hours, or days. In some embodiments more than oneconditioning step or more than one polynucleotide molecule applicationcan be performed on the same plant. In embodiments of the method, thesegment can be cloned or identified from (a) coding (protein-encoding),(b) non-coding (promoter and other gene related molecules), or (c) bothcoding and non-coding parts of the Mildew Resistance Locus O (MLO)target gene. Non-coding parts include DNA, such as promoter regions orthe RNA transcribed by the DNA that provide RNA regulatory molecules,including but not limited to: introns, 5′ or 3′ untranslated regions,and microRNAs (miRNA), trans-acting siRNAs, natural anti-sense siRNAs,and other small RNAs with regulatory function or RNAs having structuralor enzymatic function including but not limited to: ribozymes, ribosomalRNAs, t-RNAs, aptamers, and riboswitches. In certain embodiments wherethe polynucleotide used in the composition comprises a promoter sequenceessentially identical to, or essentially complementary to, at least 18contiguous nucleotides of the promoter of the endogenous target gene,the promoter sequence of the polynucleotide is not operably linked toanother sequence that is transcribed from the promoter sequence.

Compositions comprising a polynucleotide and a transfer agent providedherein can be topically applied to a plant or plant part by anyconvenient method, e.g., spraying or coating with a powder, or with aliquid composition comprising any of an emulsion, suspension, orsolution. Such topically applied sprays or coatings can be of either allor of any a portion of the surface of the plant or plant part.Similarly, compositions that comprise a transfer agent or otherpre-treatment can in certain embodiments be applied to the plant orplant part by any convenient method, e. g., spraying or wiping asolution, emulsion, or suspension. Compositions comprising apolynucleotide and a transfer agent provided herein can be topicallyapplied to plant parts that include, but are not limited to, flowers,stems, tubers, meristems, ovules, fruit, anthers, pollen, leaves, orseeds.

Application of compositions comprising a polynucleotide and a transferagent to seeds is specifically provided herein. Seeds can be contactedwith such compositions by spraying, misting, immersion, and the like.

In certain embodiments, application of compositions comprising apolynucleotide and a transfer agent to plants, plant parts, or seeds inparticular can provide for an improvement in fungal disease resistanceand/or nematode resistance in progeny plants, plant parts, or seedsderived from those treated plants, plant parts, or seeds. In certainembodiments, progeny plants, plant parts, or seeds derived from thosetreated plants, plant parts, or seeds will exhibit an improvement infungal disease resistance and/or nematode resistance that result fromsuppressing expression of an MLO gene. In certain embodiments, themethods and compositions provided herein can provide for an improvementin fungal disease resistance and/or nematode resistance in progenyplants or seeds as a result of epigenetically inherited suppression ofMLO expression. In certain embodiments, such progeny plants exhibit animprovement in fungal disease resistance and/or nematode resistance fromepigenetically inherited suppression of MLO gene expression that is notcaused by a transgene where the polynucleotide is operably linked to apromoter, a viral vector, or a copy of the polynucleotide that isintegrated into a non-native location in the chromosomal DNA of theplant. Without seeking to be limited by theory, progeny plants or seedsderived from those treated plants, plant parts, or seeds can exhibit animprovement in an improvement in fungal disease resistance and/ornematode resistance through an epigenetic mechanism that provides forpropagation of an epigenetic condition where suppression of MLO geneexpression occurs in the progeny plants, plant parts, or plant seeds. Incertain embodiments, progeny plants or seeds exhibiting an improvementin fungal disease resistance and/or nematode resistance as a result ofepigenetically inherited suppression of MLO gene expression can alsoexhibit increased methylation, and in particular, increased methylationof cytosine residues, in the endogenous MLO gene of the plant. Plantparts, including seeds, of the progeny plants that exhibit animprovement in an improvement in fungal disease resistance and/ornematode resistance as a result of epigenetically inherited suppressionof MLO gene expression, can also in certain embodiments exhibitincreased methylation, and in particular, increased methylation ofcytosine residues, in the endogenous MLO gene. In certain embodiments,DNA methylation levels in DNA encoding the endogenous MLO gene can becompared in plants that exhibit an improvement in fungal diseaseresistance and/or nematode resistance and control plants that do notexhibit an improvement in fungal disease resistance and/or nematoderesistance to correlate the presence of the an improvement in fungaldisease resistance and/or nematode resistance to epigeneticallyinherited suppression of MLO gene expression and to identify plants thatcomprise the epigenetically inherited improvement in fungal diseaseresistance and/or nematode resistance.

Various methods of spraying compositions on plants or plant parts can beused to topically apply to a plant surface a composition comprising apolynucleotide that comprises a transfer agent. In the field, acomposition can be applied with a boom that extends over the crops anddelivers the composition to the surface of the plants or with a boomlesssprayer that distributes a composition across a wide area. Agriculturalsprayers adapted for directional, broadcast, or banded spraying can alsobe used in certain embodiments. Sprayers adapted for spraying particularparts of plants including, but not limited to, leaves, the undersides ofleaves, flowers, stems, male reproductive organs such as tassels,meristems, pollen, ovules, and the like can also be used. Compositionscan also be delivered aerially, such as by a crop dusting airplane. Incertain embodiments, the spray can be delivered with a pressurizedbackpack sprayer calibrated to deliver the appropriate rate of thecomposition. In certain embodiments, such a backpack sprayer is a carbondioxide pressurized sprayer with a 11015 flat fan or equivalent spraynozzle with a customized single nozzle assembly (to minimize waste) at aspray pressure of about 0.25 MPa and/or any single nozzle sprayerproviding an effective spray swath of 60 cm above the canopy of 3 to 12inch tall growing plants can be used. Plants in a greenhouse or growthchamber can be treated using a track sprayer or laboratory sprayer witha 11001 XR or equivalent spray nozzle to deliver the sample solution ata determined rate. An exemplary and non-limiting rate is about 140 L/haat about 0.25 MPa pressure.

In certain embodiments, it is also contemplated that a plant part can besprayed with the composition comprising a polynucleotide that comprisesa transfer agent. Such plant parts can be sprayed either pre- orpost-harvest to provide for an improvement in fungal disease resistanceand/or nematode resistance in the plant part that results fromsuppression of MLO gene expression. Compositions can be topicallyapplied to plant parts attached to a plant by a spray as previouslydescribed. Compositions can be topically applied to plant parts that aredetached from a plant by a spray as previously described or by analternative method. Alternative methods for applying compositions todetached parts include, but are not limited to, passing the plant partsthrough a spray by a conveyor belt or trough, or immersing the plantparts in the composition.

Compositions comprising polynucleotides and transfer agents can beapplied to plants or plant parts at one or more developmental stages asdesired and/or as needed. Application of compositions to pre-germinationseeds and/or to post-germination seedlings is provided in certainembodiments. Seeds can be treated with polynucleotide compositionsprovided herein by methods including, but not limited to, spraying,immersion, or any process that provides for coating, imbibition, and/oruptake of the polynucleotide composition by the seed. Seeds can betreated with polynucleotide compositions using seed batch treatmentsystems or continuous flow treatment systems. Seed coating systems areat least described in U.S. Pat. Nos. 6,582,516, 5,891,246, 4,079,696,and 4,023,525. Seed treatment can also be effected in laboratory orcommercial scale treatment equipment such as a tumbler, a mixer, or apan granulator. A polynucleotide composition used to treat seeds cancontain one or more other desirable components including, but notlimited to liquid diluents, binders to serve as a matrix for thepolynucleotide, fillers for protecting the seeds during stressconditions, and plasticizers to improve flexibility, adhesion and/orspreadability of the coating. In addition, for oily polynucleotidecompositions containing little or no filler, drying agents such ascalcium carbonate, kaolin or bentonite clay, perlite, diatomaceous earthor any other adsorbent material can be added. Use of such components inseed treatments is described in U.S. Pat. No. 5,876,739. Additionalingredients can be incorporated into the polynucleotide compositionsused in seed treatments. Such ingredients include but are not limitedto: conventional sticking agents, dispersing agents such asmethylcellulose (Methocel A15LV or Methocel A15C, for example, serve ascombined dispersant/sticking agents for use in seed treatments),polyvinyl alcohol (e.g., Elvanol 51-05), lecithin (e.g., Yelkinol P),polymeric dispersants (e.g., polyvinylpyrrolidone/vinyl acetate PVPNAS-630), thickeners (e.g., clay thickeners such as Van Gel B to improveviscosity and reduce settling of particle suspensions), emulsionstabilizers, surfactants, antifreeze compounds (e.g., urea), dyes,colorants, and the like that can be combined with compositionscomprising a polynucleotide and a transfer agent. Further ingredientsused in compositions that can be applied to seeds can be found inMcCutcheon's, vol. 1, “Emulsifiers and Detergents,” MC PublishingCompany, Glen Rock, N.J., U.S.A., 1996 and in McCutcheon's, vol. 2,“Functional Materials,” MC Publishing Company, Glen Rock, N.J., U.S.A.,1996. Methods of applying compositions to seeds and pesticidalcompositions that can be used to treat seeds are described in US PatentApplication publication 20080092256, which is incorporated herein byreference in its entirety.

Application of the compositions in early, mid-, and late vegetativestages of plant development is provided in certain embodiments.Application of the compositions in early, mid- and late reproductivestages is also provided in certain embodiments. Application of thecompositions to plant parts at different stages of maturation is alsoprovided.

The following examples are included to demonstrate examples of certainpreferred embodiments of the invention. It should be appreciated bythose of skill in the art that the techniques disclosed in the examplesthat follow represent approaches the inventors have found function wellin the practice of the invention, and thus can be considered toconstitute examples of preferred modes for its practice. However, thoseof skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentsthat are disclosed and still obtain a like or similar result withoutdeparting from the spirit and scope of the invention.

EXAMPLES Example 1 Polynucleotides of the Invention Related to the MLOTarget Gene Sequences

The amino acid sequences of proteins encoded by targeted MLO genes andtranscripts are provided in Table 2 with reference to SEQ ID NOs. 1-27of the sequence listing. Such protein sequences can be used to identifyorthologous MLO genes and transcripts from other plants not providedherewith. Such orthologous genes and their transcripts can then serve astargets of polynucleotides provided herein or as a source ofpolynucleotides that are specifically designed to target the orthologousgenes or transcripts.

The target MLO polynucleotide molecule at least occurs in the genome ofplants provided in Table 2. The MLO genes provided in Table 3, or theircorresponding transcripts, can be used as targets of polynucleotidecompositions comprising a polynucleotide of at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto those genes or transcripts. The proteins and genes respectivelyprovided in Tables 2 and 3, or sequences contained within those proteinsor genes can also be used to identify orthologous MLO genes from plantsnot listed in Tables 2 and 3. Such orthologous genes and theirtranscripts can then serve as targets of polynucleotides provided hereinor as a source of polynucleotides that are specifically designed totarget the orthologous genes or transcripts.

TABLE 2 Target MLO gene protein sequences SEQ ID NO: 1 Hordeum vulgare 1MLO-like protein from barley SEQ ID NO: 2 Sorghum bicolor 2 MLO-likeprotein from sorghum SEQ ID NO: 3 Zea mays 3 MLO-like protein from cornSEQ ID NO: 4 Gossypium hirsutum MLO-like protein from cotton SEQ ID NO:5 Glycine max 3 MLO-like protein from soybeans SEQ ID NO: 6 Glycine max4 MLO-like protein from soybeans SEQ ID NO: 7 Cucumis sativus 2 MLO-likeprotein from cucumber SEQ ID NO: 8 Cucumis sativus 4 MLO-like proteinfrom cucumber SEQ ID NO: 9 Cucumis sativus 5 MLO-like protein fromcucumber SEQ ID NO: 10 Cucumis sativus 6 MLO-like protein from cucumberSEQ ID NO: 11 Vitis vinifera 1 MLO-like protein from grape SEQ ID NO: 12Vitis vinifera 7 MLO-like protein from grape SEQ ID NO: 13 Arabidopsisthaliana 3 MLO-like protein from Arabidopsis SEQ ID NO: 14 Arabidopsisthaliana 9 MLO-like protein from Arabidopsis SEQ ID NO: 15 Arabidopsisthaliana 4 MLO-like protein from Arabidopsis SEQ ID NO: 16 Arabidopsisthaliana 8 MLO-like protein from Arabidopsis SEQ ID NO: 17 Arabidopsisthaliana 7 MLO-like protein from Arabidopsis SEQ ID NO: 18 Arabidopsisthaliana 2 MLO-like protein from Arabidopsis SEQ ID NO: 19 Arabidopsisthaliana 10 MLO-like protein from Arabidopsis SEQ ID NO: 20 Arabidopsisthaliana 11 MLO-like protein from Arabidopsis SEQ ID NO: 21 Arabidopsisthaliana 6 MLO-like protein from Arabidopsis SEQ ID NO: 22 Arabidopsisthaliana 13 MLO-like protein from Arabidopsis SEQ ID NO: 23 Arabidopsisthaliana 14 MLO-like protein from Arabidopsis SEQ ID NO: 24 Arabidopsisthaliana 12 MLO-like protein from Arabidopsis SEQ ID NO: 25 Arabidopsisthaliana 1 MLO-like protein from Arabidopsis SEQ ID NO: 26 Arabidopsisthaliana 15 MLO-like protein from Arabidopsis SEQ ID NO: 27 Arabidopsisthaliana 5 MLO-like protein from Arabidopsis

TABLE 3 Target gene sequences SEQ ID NO: 28 Hordeum vulgare MLO proteinfrom barley SEQ ID NO: 29 Hordeum vulgare MLO cDNA from barley SEQ IDNO: 30 Cucumis sativus 1 MLO-like protein from cucumber SEQ ID NO: 31Cucumis sativus 1 MLO-like cDNA from cucumber SEQ ID NO: 32 Cucumissativus 3 MLO-like protein from cucumber SEQ ID NO: 33 Cucumis sativus 3MLO-like cDNA from cucumber SEQ ID NO: 34 Cucumis sativus 7 MLO-likeprotein from cucumber SEQ ID NO: 35 Cucumis sativus 7 MLO-like cDNA fromcucumber SEQ ID NO: 36 Cucumis sativus 8 MLO-like protein from cucumberSEQ ID NO: 37 Cucumis sativus 8 MLO-like cDNA from cucumber SEQ ID NO:38 Lactuca sativa MLO-like protein from lettuce SEQ ID NO: 39 Lactucasativa MLO-like cDNA from lettuce SEQ ID NO: 40 Pisum sativum 1 MLO-likeprotein from pea SEQ ID NO: 41 Pisum sativum 1 MLO-like cDNA from peaSEQ ID NO: 42 Medicago truncatula MLO-like protein from barrel cloverSEQ ID NO: 43 Medicago truncatula MLO-like cDNA from barrel clover SEQID NO: 44 Glycine max 1 MLO-like protein from soybean SEQ ID NO: 45Glycine max 1 MLO-like cDNA from soybean SEQ ID NO: 46 Glycine max 2MLO-like protein from soybean SEQ ID NO: 47 Glycine max 2 MLO-like cDNAfrom soybean SEQ ID NO: 48 Capsicum annuum MLO-like protein from pepperSEQ ID NO: 49 Capsicum annuum MLO-like cDNA from pepper SEQ ID NO: 50Solanum lycopersicum 1 MLO-like protein from tomato SEQ ID NO: 51Solanum lycopersicum 1 MLO-like cDNA from tomatos SEQ ID NO: 52 Solanumlycopersicum 2 MLO-like protein from tomato SEQ ID NO: 53 Solanumlycopersicum 2 MLO-like cDNA from tomato SEQ ID NO: 54 Triticum aestivumMLO-like protein from wheat SEQ ID NO: 55 Triticum aestivum MLO-likecDNA from wheat SEQ ID NO: 56 Triticum aestivum 1 MLO-like protein fromwheat SEQ ID NO: 57 Triticum aestivum 1 MLO-like cDNA from wheat SEQ IDNO: 58 Triticum aestivum 2 MLO-like protein from wheat SEQ ID NO: 59Triticum aestivum 2 MLO-like cDNA from wheat SEQ ID NO: 60 Vitisvinifera 17 MLO-like protein from grape SEQ ID NO: 61 Vitis vinifera 17MLO-like cDNA from grape SEQ ID NO: 62 Vitis vinifera 13 MLO-likeprotein from grape SEQ ID NO: 63 Vitis vinifera 13 MLO-like cDNA fromgrape SEQ ID NO: 64 Vitis vinifera 6 MLO-like protein from grape SEQ IDNO: 65 Vitis vinifera 6 MLO-like cDNA from grape SEQ ID NO: 66 Vitisvinifera 3 MLO-like protein from grape SEQ ID NO: 67 Vitis vinifera 3MLO-like cDNA from grape SEQ ID NO: 68 Zea mays 1 MLO-like protein fromcorn SEQ ID NO: 69 Zea mays 1 MLO-like cDNA from corn SEQ ID NO: 70Sorghum bicolor 1 MLO-like protein from sorghum SEQ ID NO: 71 Sorghumbicolor 1 MLO-like cDNA from sorghum SEQ ID NO: 72 Oryza sativa japonica1 MLO-like protein from rice SEQ ID NO: 73 Oryza sativa japonica 1MLO-like cDNA from rice SEQ ID NO: 74 Oryza sativa japonica 2 MLO-likeprotein from rice SEQ ID NO: 75 Oryza sativa japonica 2 MLO-like cDNAfrom rice SEQ ID NO: 76 Oryza sativa japonica 3 MLO-like protein fromrice SEQ ID NO: 77 Oryza sativa japonica 3 MLO-like cDNA from rice SEQID NO: 78 Oryza sativa indica MLO-like protein from rice SEQ ID NO: 79Oryza sativa indica MLO-like cDNA from rice

The sequence listing contains the target MLO DNA sequences from theindicated plant species of Table 3. For each gene having a DNA sequenceprovided in the sequence listing and listed in Table 3, polynucleotidessuch as single stranded or double stranded DNA or RNA fragments in senseand/or antisense orientation will be mixed with an organosiliconepreparation. These compositions will be topically applied to plants toeffect expression of the target genes in the specified plant to obtainthe plants that exhibit disease resistance. In particular, plants thatare resistant to powdery mildew, downy mildew, and/or rust infectionand/or nematodes will be obtained through the application of suchcompositions.

Example 2 Identification of Orthologous MLO Genes

A bootstrapped phylogenetic tree is provided in FIG. 1. The phylogenetictree shown in FIG. 1 was generated with ClustalX version 2.0.12 usingdefault parameters. First, a multiple sequence alignment was generatedwith ClustalX using the default parameters, except that iteration ateach alignment step was done. The bootstrapped phylogenetic tree wasthen assembled using the Neighbor-Joining method. The protein sequencesdisclosed in SEQ ID NOS: 1-27 are a useful basis set for determiningwhether MLO-like proteins are functional orthologs and likely to beuseful targets for suppression in order to control fungal diseases suchas powdery mildew, downy mildew and rust infections. Specifically, MLOhomologs useful for the control of powdery mildews fall into one of twoclades: 1) the Glade with A. thaliana MLO2 (SEQ ID NO: 18), such as A.thaliana MLO6 (SEQ ID NO: 21), A. thaliana MLO12 (SEQ ID NO: 24) andPisum sativum MLO1 (SEQ ID NO: 40) but not A. thaliana MLO11 (SEQ ID NO:20) or 2) in the Glade with Hordeum vulgare MLO (SEQ ID NO: 28), Zeamays MLO1 (SEQ ID NO: 68) and Sorghum bicolor MLO1 (SEQ ID NO: 70) butnot Sorghum bicolor MLO2 (SEQ ID NO: 2)

The sequences disclosed in SEQ ID NO: 1 through 79, along with thephylogentic method for functional assignment described above, can beused to efficiently identify and clone MLO homologs useful for thecontrol of pathogens causing powdery mildews, downy mildews or rusts,from other plant species not explicitly described herein.

Example 3 Polynucleotides that can be Used to Reduce MLO Expression inVarious Plants

Examples of polynucleotides that can be used to reduce expression of MLOgenes in various plants is provided herewith as SEQ ID NOS: 80-195. TheSEQ ID NOS: 80-195 describe sense/antisense double stranded RNA targetedto the coding regions of Mildew Resistance Locus O (MLO) sequences froma variety of dicot and monocot plants and are useful for downregulatingMLO expression using methods described herein. Other regions of MLOgenes can also be targeted to modify expression including coding regionsand/or promoter regions. Polynucleotides that can be used to reduce MLOexpression include sense/antisense dsRNA, antisense RNA, sense orantisense ssDNA as well as sense/antisense double stranded DNA. Forexample, a polynucleotide that comprises at least 18 contiguousnucleotides that are essentially identical or essentially complementaryto SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, or 79 can be used todownregulate expression of those MLO genes.

Example 4 Topical Polynucleotide Application and Powdery Mildew TestingMethods

Barley seeds are planted in 2 inch pots in the greenhouse. Five dayslater, barley seedlings are sprayed with polynucleotides such as ssDNAand/or dsRNA polynucleotides directed to the promoter and/or the codingregion of a target gene of interest. For example, polynucleotidesdirected to MLO include polynucleotides that comprise at least 18contiguous nucleotides that are essentially identical or complementaryto SEQ ID NO:28 or 29. Other examples of polynucleotides that target MLOfor down regulation include polynucleotides of SEQ ID NO:80 to 83. Anucleotide solution of 6-20 nm of each ssDNA or 0.5-4 nm dsRNA, 0.1 to0.3% L77 silwet, 50 mM NaPO₄ in a final volume of 40 microliters ofwater is applied. Two to 4 days post spraying, seedlings will beinfected with dry spores of barley powdery mildew (Blumeria graminis f.sp. hordei) and 7 days post infection, disease development is determinedby scoring for the percentage of leaf area covered with powdery mildew.

Cucumber seeds are planted in 3-inch square pots and thinned to oneplant per pot after emergence. When the first true leaf is fullyexpanded and the second leaf is opening, a polynucleotide solution, suchas ssDNA and/or dsRNA polynucleotides directed to the promoter and/orthe coding region of a target gene of interest, is applied to the firsttrue leaf or the cotyledons. For example, polynucleotides directed toMLO include polynucleotides that comprise at least 18 contiguousnucleotides that are essentially identical or complementary to SEQ IDNO:38 or 39. Examples of polynucleotides also include polynucleotides ofSEQ ID NO:100 to 103. A nucleotide solution of 6-20 nm of each ssDNA or0.5-4 nm dsRNA, 0.1 to 0.3% L77 Silwet, 50 mM NaPO₄ in a final volume of40 microliters of water is applied. Two days later the entire cucumberplant is inoculated with a shower of dry spores of cucumber powderymildew (Sphaerotheca fuliginea) shaken off diseased plants. Diseaseseverity will be evaluated on the treated leaf and succeeding leaves 10days later and at subsequent intervals.

Tomato seeds are planted in a 3-inch square pots and thinned to oneplant per pot after emergence. Two weeks old tomato seedlings aretreated with 6-20 nm of each ssDNA or 0.5-4 nm dsRNA, 0.2-0.5% L77silwet, 50 mM NaPO₄, 1% ammonium sulfate in a final volume of 30microliters of water. For example, polynucleotides directed to MLOinclude polynucleotides that comprise at least 18 contiguous nucleotidesthat are essentially identical or complementary to SEQ ID NO:50, 51, 52,or 53. Examples of polynucleotides also include polynucleotides of SEQID NO:126 to 131. Two to 4 days post spraying plants are inoculated withdry spores of tomato powdery mildew (Oidium neolycopersici) and 13 dayspost infection, disease development is scored for the percentage of leafarea covered with powdery mildew.

Example 5 Protection of Barley from Powdery Mildew by TopicalPolynucleotide Application

Barley seeds were planted in 2 inch pots in the greenhouse. Five dayslater, barley seedlings were sprayed with the indicated polynucleotidesor a control formulation according to either the Treatment 1 orTreatment 2 methods of Tables 4 and 5, respectively. The liquids appliedto the plants are provided in Table 6 and the description of nucleicacid sequences of the ssDNA polynucleotides used is provided in Table 7.Post spraying, the seedlings were infected with dry spores of barleypowdery mildew (Blumeria graminis f. sp. hordei) and 7 days postinfection, disease development was scored for the percentage of leafarea covered with powdery mildew. The average leaf length was alsoscored. Results of this analysis are shown in FIG. 2.

TABLE 4 Treatment 1 Treatment 1 5 mM NaPO4 0.3% Silwet 2 × 5 ul/leafDisease infection 3 days later

TABLE 5 Treatment 2 Treatment 2 5 mM NaPO4 1% AMS 0.2% Silwet 2 × 7.5ul/leaf Disease infection 2 day later

TABLE 6 Liquids Tested Liquid trigger type nucleotide ID # conc silwetTreatment # S Formulation 0.30% first leaf Treatment 1 C DNA GFP GFP_as1X12 nmol 0.30% first leaf Treatment 1 1 DNA mlo T4213as, T4214as,T4215as 3X12 nmol 0.30% first leaf Treatment 1 2 DNA mlo T4216as,T4217as, T4218as 3X12 nmol 0.30% first leaf Treatment 1 3 DNA mloT4211as, T4219as, T4220as 3X36 nmol 0.20% first leaf Treatment 2 4 DNAmlo T4212as, T4214as, T4222as 3X36 nmol 0.20% first leaf Treatment 2 5Formulation 0.20% first leaf Treatment 2 6 DNA GFP GFP_as 1X36 nmol0.20% first leaf Treatment 2

TABLE 7 Polynucleotides used Target Plant and SEQ Gene Type NameSequence ID Length Target barley antisense T4211_AS GGGGTGCT 184 24 ORFMLO DNA GGAGAGGC CCAGGTGG barley antisense T4212_AS CGACGTCT 185 23 ORFMLO DNA GGTGCGTG AACCGGA barley antisense T4213_AS CTGGTATT 186 23 ORFMLO DNA CCAAGGAG GTGGTCT barley antisense T4214_AS GATGAGGA 187 23 ORFMLO DNA GCAGGGAT ATGAAGC barley antisense T4215_AS ATGAGCTC 188 23 ORFMLO DNA CGCCTTCA TCTTCTC barley antisense T4216_AS GGCCTTCT 189 23 ORFMLO DNA TGTGCCGG TGCTGGA barley antisense T4217_AS CTGTCCAC 190 23 ORFMLO DNA ACAAAATG CGCCATC barley antisense T4218_AS GTTCTGGA 191 22 ORFMLO DNA ACAACGTC AGGTGT barley antisense T4219_AS GTCGGGGC 192 22 ORFMLO DNA GGTGGAAC CAGAAG barley antisense T4220_AS AAAAATCT 193 21 ORFMLO DNA GCACTGGG GATGT barley antisense T4221_AS GATTTAGT 194 24 ORF MLODNA CTGTGCAC CGGGTGCG barley antisense T4222_AS AACCGGGT 195 24 ORF MLODNA ACATGTCC CTAGCCTC

FIG. 2 shows that the percentage disease area was significantly deceasedin plants treated with Silwet formulations containing the barley MLOantisense DNA polynucleotides relative to both the Silwet formulationalone or the Silwet formulation combined with a control GFP (GreenFluorescent Protein) polynucleotide (SEQ ID NO:196).

Example 6 Protection of Barley from Powdery Mildew by TopicalPolynucleotide Application

Barley seeds were planted in 2 inch pots in the greenhouse. Five dayslater, barley seedlings were sprayed with the indicated polynucleotidesor a control formulation according to either the Treatment 1 orTreatment 2 methods of Tables 8 and 9, respectively. The liquids appliedto the plants are provided in Table 10 and the description of nucleicacid sequences of the ssDNA polynucleotides used is provided in Table 7.Post spraying, the seedlings were infected with dry spores of barleypowdery mildew (Blumeria graminis f. sp. hordei) and 7 days postinfection, disease development was scored for the percentage of leafarea covered with powdery mildew. The average leaf length was alsoscored. Results of this analysis are shown in Table 11.

TABLE 8 Treatment 1 Treatment 1 5 mM NaPO4 1% AMS 0.25% Silwet 2 × 5 ulDisease infection 2 days later

TABLE 9 Treatment 2 Treatment 2 5 mM NaPO4 1% AMS 0.25% Silwet 2 × 6 ulDisease infection 1 day later

TABLE 10 Liquids Tested Liq- trigger Concentration of uid typepolynucleotide ID # polynucleotides Treatment # 0 0 Treatment 1 7 DNAGFP GFP_as 40 nmol Treatment 1 8 DNA GFP GFP_as 80 nmol Treatment 1 9DNA mlo, T4211as,13, 15, 22 4 × 10 nmol Treatment 1 antisense 10 DNAmlo, T4212as, T4219as, 4 × 20 nmol Treatment 1 antisense T4220as,T4221as 11 DNA GFP GFP_as 48 nmol Treatment 2 12 DNA GFP GFP_as 96 nmolTreatment 2 13 DNA mlo, T4211as, T4215as, 4 × 12 nmol Treatment 2antisense T4216as, T4218as 14 DNA mlo, T4219as, T4220as, 4 × 24 nmolTreatment 2 antisense T4221as, T4222as 15 0 Treatment 2

Table 11 shows that the percentage disease area was significantlydecreased in plants treated with Silwet formulations containing thebarley MLO antisense DNA polynucleotides relative to both thenon-treated control or the Silwet formulation combined with a controlGFP (Green Fluorescent Protein) polynucleotide.

TABLE 11 Percentage of disease area trigger Liquid type R1 R2 R3 R4 R5R6 avr stdev NT NT 10 25 10 5 13 8.7 15 Blank 25 5 5 5 10 10 11 DNA 5 510 5 1 10 6 3.5 GFP 12 DNA 5 1 1 5 5 3.4 2.2 GFP 13 DNA 1 1 1 1 1 5 1.71.6 MLO 14 DNA 1 1 1 5 1 1.8 1.8 MLO  7 DNA 25 10 10 10 10 10 12.5 7.1GFP  8 DNA 10 5 5 5 10 5 3.5 GFP  9 DNA 5 1 1 5 1 2.6 2.1 MLO 10 DNA 5 55 5 1 4.1 1.7 MLO R1-R6 are replicates 1 through 6.

Example 7 Topical Polynucleotide Application and Nematode TestingMethods

Application of Polynucleotides to Leaves for Nematode Control

Ten day old cucumber plants grown in sand are spotted with nucleotides,either ssDNA and/or dsRNA polynucleotides, directed to the promoterand/or the coding region of a target gene of interest. A nucleotidesolution of 6-20 nm of each ssDNA or 0.5-1 nm dsRNA, 0.1% L77 silwet, 50mM NaPO4 in a final volume of 40 uL water is applied. Two cotyledon orleaves are spotted with 20 uL of the nucleotide solution for a total of40 uL per plant. After 6-24 hours, 1000 vermiform eggs or 1000 J2Meloidogyne incognita (RKN) are inoculated into each pot. Watering ofthe test plants is then restricted to only water as needed to preventwilt for a period of 24 hours. After the 24 hour restricted watering,normal sub-irrigation watering is done for the duration of the test.Cucumber plants are harvested approximately 14 days after inoculation bywashing sand off the roots. A root gall rating and visual phytotoxicityrating is assigned using the following scales: Gall rating scale (Gall:% root mass galled): 0=0-5%; 1=6-20%; 2=21-50%; and 3=51-100%. Visualphytotoxicity scale is also assigned (Vis. tox; visual reduction in rootmass compared to the control): rs1=mild stunting; rs2=moderate stunting;rs3=severe stunting.

Experiments in soybeans using soy cyst nematodes (SCN) are similar tothe cucumber RKN assay except for the following changes. Soybean seedsare planted in 100% sand in two inch square plastic pots. Thepolynucleotide solution is applied when the soybeans show the firsttrifoliate beginning to emerge, about 10 to 12 days after planting. Atleast six hours after application of the polynucleotide solution, thenematode soybean cyst nematode (SCN) inoculum (1000 vermiform eggs or1000 J2s) is applied to the pots. Watering of the test plants is thenrestricted to only water as needed to prevent wilt for a period of 24hours. After the 24 hour restricted watering, normal sub-irrigationwatering is done for the duration of the test. Twenty eight days afterinoculation the plants are harvested and cysts counted.

Experiments in corn using lesion nematodes are similar to above exceptfor the following changes. Corn plants growing in a sand:Turface mix 2:1in 4 inch deep pots (Turface™ MVP, Profile Products, LLC., BuffaloGrove, Ill.). Treatment with polynucleotide solution is done when theplants are approximately 8-10 days old. At least six hours afterinoculation of the polynucleotide solution, plants are inoculated with 2gm of P. scribneri infested corn roots which are then removed from thepot after 7 days. Watering of the test plants is then restricted to onlywater as needed to prevent wilt for a period of 24 hours afterinoculation. After the 24 hour restricted watering, normalsub-irrigation watering as needed is done for the duration of the test.12-14 days post inoculation, plants are harvested and nematodesextracted for 6 days from the cut up roots in a mist tent.

Application of Polynucleotides to Seeds for Nematode Control

Cucumber seeds are soaked approximately 5-72 hours in nucleotides,either ssDNA and/or dsRNA polynucleotides directed to the promoterand/or targeting the coding region of a target of interest. Seeds canalso be soaked in water for a few hours prior to soaking inpolynucleotide solution. Soaking solution consists of 20 nm of eachssDNA or 0.03-1 nm dsRNA, 0.1% silwet L77, 50 mM NaPO4 in a final volume200 uL in water. The radicals of the cucumber seeds emerge within 72hours, after which the seeds are placed on germination paper until rootlength is approximately 2 inches. Seedlings are transplanted to sandvials for RKN inoculation 24 hours later. Ten mL dry sand is added toeach vial and seedlings are planted by tilting the vial and laying theseedling in the correct orientation so that the cotyledons are justabove the sand and then tilting back to cover the radicles with sand.3.3 ml water is added to each vial and the vials placed in racks underfluorescent light banks. 500 vermiform eggs or 300 J2 RKN are inoculatedin each tube in 50 uL of deionized or spring water. Harvest of thecucumber plants is performed 10 to 12 days after inoculation by washingsand off the roots. A root gall rating and visual phytotoxicity ratingis assigned using the following scales: Gall rating scale (Gall: % rootmass galled): 0=0-5%; 1=6-20%; 2=21-50%; and 3=51-100%. The average ofthe triplicate gall rating is then calculated: green=0.00-0.33 (nogalls); yellow=0.67-1.33 (mild galling); orange=1.67-2.33 (moderategalling); red=2.67-3.00 (severe galling). Visual phytotoxicity scale isalso assigned (Vis. tox; visual reduction in root mass compared to thecontrol): rs1=mild stunting; rs2=moderate stunting; rs3=severe stunting.

Experiments in soybeans using soy cyst nematodes (SCN) are similar toRKN assays except for the following changes. After 5-72 hours of soakingsoybean seeds are planted in 100% sand in two inch square plastic pots.Seeds can also be soaked in water for a few hours prior to soaking inpolynucleotide solution. Seven days after planting the soybean seed, thenematode soybean cyst nematode (SCN) inoculum (1000 vermiform eggs or1000 J2s) are applied to the pot. Watering of the test plants is thenrestricted to only water as needed to prevent wilt for a period of 24hours. After the 24 hour restricted watering, normal sub-irrigationwatering is done for the duration of the test. Twenty eight days afterinoculation the test is harvested and cysts counted.

Experiments in corn using lesion nematodes are similar to above exceptfor the following changes. After 5-72 hours of soaking corn seeds areplanted in a sand:turface mix 2:1 in 4 inch deep pots. Seeds can also besoaked in water for a few hours prior to soaking in polynucleotidesolution. Inoculum of 2 gm of roots P. scribneri infested corn roots areapplied at seeding and removed from the pot after 7 days. Watering ofthe test plants is then restricted to only water as needed to preventwilt for a period of 24 hours after inoculation. After the 24 hourrestricted watering, normal sub-irrigation watering as needed is donefor the duration of the test. 12-14 days post inoculation, plants areharvested and nematodes extracted for 6 days from the cut up roots in amist tent.

RKN and SCN J2s are prepared from hatchbowls using the followingsolutions: RKN solution: 1 L aerated tap water, 1 ml of 50 mg/mlkanamycin, 0.5 ml of 20 mg/ml imazalil sulfate; SCN solution: 1 Laerated tap water, 1 ml of 50 mg/ml kanamycin, 0.5 ml of 20 mg/mlimazalil sulfate, 1430 mg zinc sulfate.

Hatchbowls are autoclaved 6 oz bowls, lined with screen mesh and paperfilter. Approximately 20 ml of appropriate hatch solution is poured intoeach bowl. Eggs are then place in the bowls and covered with foil. Thebowls are then placed in a 25° C. incubator overnight. The next day thehatched J2's are extracted, additional solution added as needed andreplaced in the incubator. Each bowl is used for 2 weeks and thendisposed.

Example 8 Protection of Barley from Powdery Mildew by TopicalPolynucleotide Application

Barley seeds were planted in 2 inch pots in the greenhouse. Five dayslater, barley seedlings were sprayed with the indicated polynucleotidesor a control formulation according to the methods of Table 12. Thedescription of nucleic acid sequences of the ssDNA polynucleotides usedis provided in Table 13. Post spraying, the seedlings were infected withdry spores of barley powdery mildew (Blumeria graminis f sp. hordei) and7 days post infection, disease development was scored for the percentageof leaf area covered with powdery mildew. Results of these experimentsare shown in Table 14, ANOVA statistical calculations are shown in Table15 and a corresponding graph with LSD bar is shown in FIG. 3.

TABLE 12 Treatment Protocols Trt trigger type nucleotide ID # rationmol/plant* 1 DNA GFP GFP 13.4 2 DNA mlo T4211 13.4 3 DNA mlo T4219 13.44 DNA mlo T4221 13.4 5 DNA mlo T4211/T4219 1 to 1 6.7/6.7 6 DNA mloT4211/T4221 1 to 1 6.7/6.7 7 DNA mlo T4219/T4221 1 to 1 6.7/6.7 8 DNAmlo T4211/19as/21 1 to 1 to 1 4.5 each 9 DNA mlo T4211/T4221 1 to 33.3/10  10 DNA mlo T4211/GFP 1 to 1 6.7/6.7 11 DNA mlo T4211/TGFP 1 to 33.3/10  12 DNA mlo T4211 3.3/11  *The indicated amounts ofpolynucleotides were provided in 5 mM NaPO₄, 1% Ammonium Sulfate, and0.25% Silwet ™ (wt percent).

TABLE 13 Polynucleotides used # Seq Sequence nucle- SEQ Name Compositionotides ID NO Organism T4211 GGGGTGCTGGAGA 24 184 barley GGCCCAGGTGGT4212 CGACGTCTGGTGC 23 185 barley GTGAACCGGA T4213 CTGGTATTCCAAG 23 186barley GAGGTGGTCT T4214 GATGAGGAGCAGG 23 187 barley GATATGAAGC T4215ATGAGCTCCGCCT 23 188 barley TCATCTTCTC T4216 GGCCTTCTTGTGC 23 189 barleyCGGTGCTGGA T4217 CTGTCCACACAAA 23 190 barley ATGCGCCATC T4218GTTCTGGAACAAC 22 191 barley GTCAGGTGT T4219 GTCGGGGCGGTGG 22 192 barleyAACCAGAAG T4220 AAAAATCTGCACT 21 193 barley GGGGATGT T4221 GATTTAGTCTGTG24 194 barley CACCGGGTGCG T4222 AACCGGGTACATG 24 195 barley TCCCTAGCCTCGFP GTTGTAGTTGTAC 25 196 A. TCCATCTTATTG victoria

TABLE 14 Percent Leaf Infection Area Results Average Percent InfectionTreatment No. nucleotide ID # Area STDEV N Variance NT 28.6 9.45 7 89.29Formulation 17.5 8.22 6 67.50 1 GFP 10.6 6.23 8 38.84 2 T4211 1.38 2.268 5.13 4 T4219 2.33 2.07 6 14.17 5 T4221 4.16 2.04 6 4.27 6 14211/T42192.2 2.59 6 4.17 1:1 7 T4211/T4221 2.2 2.59 5 6.70 1:1 8 T4219/T4221 4.21.79 5 6.70 1:1 10 T4211/T4221 2.4 2.4 5 3.20 1:3 11 T4211/GFP 1:1 2.42.4 5 4.30 12 T4211/TGFP 2.6 2.19 5 5.80 1:3 T4211 1.75 2.22 5 5.80

TABLE 15 ANOVA ANOVA Source of Variation SS df MS F P-value F critBetween 5351.475 15 356.765 17.96064 7.29E− 1.581213 Groups 19 Within1469.914 74 19.86371 Groups Total 6821.389 89

Example 9 Topical Application of Short dsRNA Polynucleotides ProvidesProtection of Barley from Powdery Mildew

Barley seeds were planted in 2 inch pots in the greenhouse. Five dayslater, barley seedlings were treated by hand application of dsRNApolynucleotides or a control formulation as indicated in Table 16.1.7-6.7 nm of the dsRNA polynucleotide, as indicated in Table 16, wasprovided in 5 mM NaPO₄, 1% Ammonium Sulfate, and 0.25% L77-Silwet™ (wtpercent). Two cotyledon or leaves were spotted with 20 uL of thenucleotide solution for a total of 40 uL per plant. The nucleotidesequence of the dsRNA polynucleotides is provided in Table 17. Postapplication, the seedlings were infected with dry spores of barleypowdery mildew (Blumeria graminis f. sp. hordei) and disease developmentwas scored at both 7 days and 14 days post infection for the percentageof leaf area covered with powdery mildew. Results of these experimentsare shown in Table 18, ANOVA statistical calculations are shown in Table19 and corresponding graph with LSD bar is shown in FIG. 4. FIG. 5 showsthe results in graph format for 14 days post-infection.

TABLE 16 Treatment Protocols for dsRNA polynucleotides Trt Trigger typeNucleotide ID# nmol/plant* NT None F None 1 dsRNA T4211_dsRNA 6.7 2dsRNA T4114_dsRNA 6.7 3 dsRNA T4217_dsRNA 6.7 4 dsRNA T4219_dsRNA 6.7 5dsRNA T4220_dsRNA 6.7 6 dsRNA T5910_dsRNA 6.7 7 dsRNA T5917_dsRNA 6.7 8dsRNA T5923_dsRNA 6.7 9 dsRNA M4214_7_9: a mix 2.2 each of T4214, T4217,T4219 10 dsRNA T4220_5910_17_23: 1.7 each a mix of T4220, T5910, T5917,and T5923 11 dsRNA GFP_dsRNA 6.7 *The indicated amounts ofpolynucleotides were provided in 5 mM NaPO₄, 1% Ammonium Sulfate, and0.25% Silwet ™ (wt percent).

TABLE 17 dsRNA polynucleotides used # SEQ Sequence nucle- ID Seq NameComposition otides NO Organism T4211_dsRNA GGGGUGCUGGAGA 24 197 barleyGGCCCAGGUGG T4114_dsRNA GAUGAGGAGCAGG 23 198 barley GAUAUGAAGCT4217_dsRNA CUGUCCACACAAA 23 199 barley AUGCGCCAUC T4219_dsRNAGUCGGGGCGGUGG 22 200 barley AACCAGAAG T4220_dsRNA AAAAAUCUGCACU 21 201barley GGGGAUGU T5910_dsRNA CGCCUUCAUCUUC 25 202 barley UCCAGCGCCUCCT5917_dsRNA GUCGUCCUCCAUC 25 203 barley GACCUCUUGAUG T5923_dsRNAUCAGCCCGAUCUG 25 204 barley CGUGUGGUAGCA GFP_dsRNA GUUGUAGUUGUAC 25 205A. UCCAUCUUAUUG victoria

TABLE 18 Percent Infection Area on treated plants Average PercentInfection Variance Treatment No. nucleotide ID # Area STDEV N (LSD) 1T4211_dsRNA 0.285714286 0.487950036 7 3 2 T4114_dsRNA 3.8571428573.484660262 7 3 3 T4217_dsRNA 3.285714286 2.138089935 7 3 4 T4219_dsRNA8.71428571 7.846746368 7 3 5 T4220_dsRNA 9.428571429 7.678045386 7 3 6T5910_dsRNA 6.285714286 8.920281866 7 3 7 T5917_dsRNA 5.1428571432.60950643 7 3 8 T5923_dsRNA 7.428571429 8.323804075 7 3 9 T4214, T4217,T4219 12 7.582875444 5 3 all dsRNA 10 T4220, T5910, T5917, 107.745966692 6 3 T5923 all dsRNA 11 GFP_dsRNA 13.57142857 8.017837257 7 3

Table 18 shows the percentage disease area was significantly reduced inplants treated with the Silwet formulation containing the barley MLOdsRNA triggers.

TABLE 19 ANOVA Source of Variation SS df MS F P-value F crit Between5242.448052 12 436.870671 9.63 5.29233E−11 1.63 Groups Within3403.142857 75  45.3752381 Groups Total 8645.590909 87

FIG. 4 shows that at six days post infection, of the eight dsRNAtriggers tested, six showed a significant reduction in the percentdisease area when compared to the GFP dsRNA control (dsR-4211, dsR-4217,dsR-4219, dsR5910, dsR-5917, and dsR5923). Similarly, as shown in FIG.5, at thirteen days post infection, four dsRNA treatments showedsignificant disease reduction when compared to the GFP dsRNA trigger(dsR-4211, dsR-4217, dsR-5910 and dsR-5923). The most efficacioustrigger appeared to be T4211_dsRNA, similar to the results obtainedusing T4211_ssDNA, the dsRNA form of this polynucleotide reduced mildewinfection by about 96%.

Example 10 Topical Application of Long dsRNA Polynucleotides ProvidesProtection of Barley from Powdery Mildew

Barley seeds were planted in 2 inch pots in the greenhouse. Five dayslater, barley seedlings were treated by hand application with dsRNApolynucleotides or a control formulation as indicated in Table 20.13.5-28 pmol of dsRNA polynucleotide as indicated in Table 20 wasprovided in 5 mM NaPO₄, 1% Ammonium Sulfate, and 0.25% L77-Silwet™ (wtpercent). Two cotyledon or leaves were spotted with 20 uL of thenucleotide solution for a total of 40 uL per plant. The description ofthe nucleotide sequences of the dsRNA polynucleotides is provided inTable 21. Following application of the dsRNA polynucleotides, theseedlings were infected with dry spores of barley powdery mildew(Blumeria graminis f. sp. hordei) and disease development was scored at6 days post infection for the percentage of leaf area covered withpowdery mildew. Results of these experiments are shown in Table 22,ANOVA statistical calculations are shown in Table 23 and correspondinggraph with LSD bar is shown in FIG. 5.

TABLE 20 Treatment Protocol for long dsRNA polynucleotides Trt Triggertype Nucleotide ID# pmol/plant* NT None F None 1 Long dsRNA 5946_150a18.6 2 Long dsRNA 5947_150a 18.6 3 Long dsRNA 5948_150a 18.6 4 LongdsRNA 5946_100a 28 5 Long dsRNA 5947_100a 28 6 Long dsRNA 5948_100a 28 7Long dsRNA 5946_80b 16.9 8 Long dsRNA dsGFP_100b 28 9 Long dsRNAdsGFP_100b 13.5

TABLE 21 Polynucleotides used # SEQ nucle- ID Seq Name Seq Compositionotides NO Organism 5946_150a AUGUCGGACAAAAAAGGGGUG 150 206 BarleyCCGGCGCGGGAGCUGCCGGAG ACGCCGUCGUGGGCGGUGGCG GUGGUCUUCGCCGCCAUGGUGCUCGUGUCCGUCCUCAUGGAA CACGGCCUCCACAAGCUCGGC CAUUGGUUCCAGCACCGGCAC AAG5947_150a CGUCGUCGGCCCUCGAAGCCG 150 207 Barley ACAUCCCCAGUGCAGAUUUUUCCUUCAGCCAGGGAUGAGACA AGUUUCUGUAUUCAUGUUAGU CCCAAUGUAUAGCCAACAUAGGAUGUGAUGAUUCGUACAAUA AGAAAUACAAUUUUUUACUGA GUC 5948_150aUGGUGGUGGGGCUAGCUCUCC 150 208 Barley AGUUCCUCUGCAGCUAUAUGACCUUCCCCCUCUACGCGCUCG UCACACAGAUGGGAUCAAACA UGAAGAGGUCCAUCUUCGACGAGCAGACGUCCAAGGCGCUCA CCAACUGGCGGAACACGGCCA AGG 5946_100aGUGGGCGGUGGCGGUGGUCUU 100 209 Barley CGCCGCCAUGGUGCUCGUGUCCGUCCUCAUGGAACACGGCCU CCACAAGCUCGGCCAUUGGUU CCAGCACCGGCACAAG 5947_100aCAGGGAUGAGACAAGUUUCUG 100 210 Barley UAUUCAUGUUAGUCCCAAUGUAUAGCCAACAUAGGAUGUGAU GAUUCGUACAAUAAGAAAUAC AAUUUUUUACUGAGUC 5948_100aCUCUACGCGCUCGUCACACAG 100 211 Barley AUGGGAUCAAACAUGAAGAGGUCCAUCUUCGACGAGCAGACG UCCAAGGCGCUCACCAACUGG CGGAACACGGCCAAGG 5946_80bUCGCCGCCAUGGUGCUCGUGU  80 212 Barley CCGUCCUCAUGGAACACGGCCUCCACAAGCUCGGCCAUUGGU UCCAGCACCGGCACAAG dsGFP_100a UCAAGGAGGAUGGCAACAUCC100 213 A. UGGGCAAUAAGAUGGAGUACA victoria ACUACAACGCCCACAAUGUGUACAUCAUGACCGACAAGGCCA AGAAUGGCAUCAAGGUGAACU UCAAGAUCCGCCACAACAUCGAGGAUGGCAGCGUGCAGCUGG CCGAC

TABLE 22 Percent Leaf Infection Area Results Avg Percent Trt No.Nucleotide ID N Infection STDEV LSD NT 8 23.125 5.303301 2.82Formulation 8 19.375 7.763238 2.82 1 dsGFP_100a 8 13.125 7.529703 2.82 25946_150a 9 5.777778 3.562926 2.82 3 5947_150a 9 9 6.819091 2.82 45948_150a 9 8.444444 6.930208 2.82 5 5946_100a 8 10 6.546537 2.82 65947_100a 9 12.77778 7.120003 2.82 7 5948_100a 9 12.22222 7.546154 2.828 dsGFP_100b 9 12.77778 7.120003 2.82 9 5946_80b 8 13.75 6.943651 2.82

TABLE 23 ANOVA Source of Variation SS df MS F P-value F crit BetweenGroups 2013.068853 10 201 4.4 0 1.7 Within Groups 3777.569444 83 46Total 5790.638298 93

FIG. 5 shows that at six days post infection of the eight dsRNAs tested,T5946 (150-mer) had significantly lower infection compared to the GFPdsRNA control. The other two 150-mer dsRNAs (T5947 and T5948) alsotrended lower compared to the control.

What is claimed is:
 1. A method for producing a barley plant exhibitingan improvement in fungal disease resistance comprising topicallyapplying to a barley plant surface a composition that comprises: a. atleast one dsRNA of 24 to about 95 nucleotides in length, wherein thedsRNA comprises a segment of 24 contiguous nucleotides that areidentical or fully complementary to SEQ ID NO: 184, wherein said dsRNAis not operably linked to a promoter or to a viral vector and whereinsaid dsRNA does not become integrated into the plant chromosome; and b.a transfer agent comprising an organosilicone preparation thatconditions the barley plant surface to permeation by the dsRNA moleculesinto plant cells, wherein said plant exhibits an improvement in fungaldisease resistance in comparison to a control barley plant that has notbeen treated with a composition comprising the dsRNA and a transferagent, wherein the fungal disease resistance results from suppression ofa barley Mildew Resistance Locus O (MLO) gene.
 2. The method of claim 1,wherein said dsRNA is not physically bound to a biolistic particle. 3.The method of claim 1, wherein said dsRNA is not transcribed from DNAintegrated into a chromosome of the plant.
 4. The method of claim 3,wherein said dsRNA is 24 nucleotides in length.
 5. The method of claim1, wherein said composition further comprises an additionalpolynucleotide of at least 24 nucleotides in length that is identical orcomplementary to SEQ ID NO:
 29. 6. A composition comprising at least onedsRNA of 24 to about 95 nucleotides in length, wherein the dsRNAcomprises a segment of 24 nucleotides that are identical or fullycomplementary to SEQ ID NO: 184, wherein said dsRNA is not operablylinked to a promoter or a viral vector; and, b) a transfer agentcomprising an organosilicone preparation that conditions a plant surfaceto permeation by the dsRNA molecules into plant cells.
 7. Thecomposition of claim 6, wherein said dsRNA is 24 nucleotides in length.8. The composition of claim 6, wherein said composition furthercomprises a non-polynucleotide herbicidal molecule, a polynucleotideherbicidal molecule, a polynucleotide that suppresses an herbicidetarget gene, an insecticide, a fungicide, a nematocide, or a combinationthereof.
 9. The composition of claim 6, wherein said dsRNA is notphysically bound to a biolistic particle.